Anti-human Respiratory Syncytial Virus (RSV) antibodies and methods of use

ABSTRACT

Provided herein are antibodies or antigen-binding fragments thereof that immunospecifically bind to the fusion (F) protein of Respiratory Syncytial Virus (RSV). Also provided are methods for of prevention, treatment and diagnosis of viral infection and/or the treatment of one more symptoms of RSV-mediated disease. Methods of generating antibodies that immunospecifically bind RSV F protein also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 14/297,486, filed Jun. 5, 2014, U.S. Pat. No. 9,403,900, whichis a continuation of U.S. patent application Ser. No. 13/809,397, filedMar. 25, 2013, U.S. Pat. No. 9,139,642, issued Sep. 22, 2015, whichapplication is a national phase entry under 35 U.S.C. § 371 ofInternational Patent Application PCT/US2011/043463 filed on Jul. 8,2011, designating the United States of America and published in Englishas International Patent Publication WO 2012/006596 A2 on Jan. 12, 2012,which claims the benefit under Article 8 of the Patent CooperationTreaty and under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication Ser. No. 61/399,310, filed Jul. 9, 2010, and to U.S.Provisional Patent Application Ser. No. 61/456,454 filed Nov. 5, 2010,the disclosure of all of which is hereby incorporated herein in itsentirety by this reference.

This application is related to U.S. application Ser. No. 12/806,498,filed Aug. 13, 2010, published as US-2011-0076268 A1, and entitled“ANTIBODIES AGAINST HUMAN RESPIRATORY SYNCTIAL VIRUS (RSV) AND METHODSOF USE,” now U.S. Pat. No. 8,568,719, issued Oct. 29, 2013, andInternational Application No. PCT/US10/045549, filed Aug. 13, 2010,published as International PCT Application No. WO 2011/020079 andentitled “ANTIBODIES AGAINST HUMAN RESPIRATORY SYNCTIAL VIRUS (RSV) ANDMETHODS OF USE,” both of which claim priority to U.S. ProvisionalApplication Ser. No. 61/274,395, filed Aug. 13, 2009, entitled“ANTIBODIES AGAINST HUMAN RESPIRATORY SYNCTIAL VIRUS (RSV) AND METHODSOF USE.” The subject matter of each of the above-referenced applicationsis incorporated by reference in its entirety.

STATEMENT ACCORDING TO 37 C.F.R. § 1.821(C) OR (E)—SEQUENCE LISTINGSUBMITTED AS PDF FILE WITH A REQUEST TO TRANSFER CRF FROM PARENTAPPLICATION

Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing a PDF versionof the Sequence Listing has been submitted concomitant with thisapplication, the contents of which are hereby incorporated by reference.The transmittal documents of this application include a Request toTransfer CRF from the parent application.

TECHNICAL FIELD

Provided are antibodies and antigen-binding fragments thereof thatimmunospecifically bind to the F protein of Respiratory Syncytial Virus(RSV) and/or to RSV and/or neutralize RSV. Also provided are diagnosticand therapeutic methods that employ anti-RSV antibodies andantigen-binding fragments thereof. The therapeutic methods includeadministering the provided anti-RSV antibodies or antigen-bindingfragments thereof for the prevention or treatment of a RSV infectionand/or amelioration of one or more symptoms of a RSV infection, such asinfections in infants and infections associated with organtransplantation. Combinations of a plurality of different anti-RSVantibodies and antigen-binding fragments thereof provided herein and/orwith other anti-RSV antibodies and antigen-binding fragments thereof canbe used for combination therapy. Compositions containing the mixtures ofanti-RSV antibodies and antigen-binding fragments thereof also areprovided.

BACKGROUND

RSV is the leading cause of severe respiratory illness in infants andyoung children and is the major cause of infantile bronchiolitis(Welliver (2003) J Pediatr 143:S112). An estimated 64 million cases ofrespiratory illness and 160,000 deaths worldwide are attributable to RSVinduced disease. In the United States alone, tens of thousands of infanthospitalizations are due to infections by paramyxoviruses, such as RSVand parainfluenza virus (PIV) (Shay et al. (1999) JAMA 282:1440-1446).Severe RSV infection occurs most often in children and infants,especially in premature infants. Underlying health problems such aschronic lung disease or congenital heart disease can significantlyincrease the risk of serious illness. RSV infections also can causeserious illness in the elderly, individuals with chronic pulmonarydisease and immunocompromised adults, such as bone marrow transplantrecipients.

Several approaches to the prevention and treatment of RSV infection havebeen investigated, including vaccine development, antiviral compounds(ribavirin), antisense drugs, RNA interference technology, and antibodyproducts, such as immunoglobulin or intravenous monoclonal antibodies.Intravenous immunoglobulin (RSV-IGIV; RESPIGAM®) isolated from donorsand a monoclonal antibody, palivizumab (SYNAGIS™), have been approvedfor RSV prophylaxis in high risk children. A vaccine or commerciallyavailable treatment for RSV, however, is not yet available. Onlyribavirin is approved for treatment of RSV infection. In order to beeffective for treatment of RSV infection, high doses, frequentadministrations and/or volumes of antibody products, such as RSV-IG andpalivizumab, are required due to low specificity. Further, the use ofproducts, such as intravenous immunoglobulin, is dependent on donoravailability. Accordingly, there exists a need for additional agents forthe prevention or treatment of RSV infections.

SUMMARY

Provided herein are isolated polypeptides, antibodies or antigen-bindingfragments thereof for the prophylaxis and treatment of RSV infection andRSV-mediated diseases or conditions. Also provided herein are isolatedpolypeptides, antibodies or antigen-binding fragments thereof for thediagnosis and/or monitoring of RSV infection. Provided herein areisolated polypeptides, antibodies or antigen-binding fragments thereofthat immunospecifically bind to and neutralize RSV. In some examples,the polypeptides provided herein immunospecifically bind to andneutralize RSV when the polypeptide provided herein is contained in anantibody or antigen-binding fragment. Also provided herein areantibodies and antigen-binding fragments that contain a polypeptideprovided herein where the antibody or antigen-binding fragmentimmunospecifically binds to and neutralizes RSV. The polypeptides,antibodies and antigen-binding fragments provided herein canspecifically bind to the F protein as well as neutralize RSV. Providedherein are isolated polypeptides, antibodies or antigen-bindingfragments thereof that can neutralize RSV subtypes A and B. Providedherein are isolated polypeptides, antibodies or antigen-bindingfragments thereof that immunospecifically bind the F protein of RSV.

Provided herein are antibodies or antigen-binding fragments thereof thatcontain a V_(H) CDR1 comprising the peptide of SEQ ID NOS:405, 411, 417,423, 429, 437-441, 458, 464, 470 or 482-484 or a sequence of amino acidshaving at least or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS:405, 411,417, 423, 429, 437-441, 458, 464, 470 or 482-484; a V_(H) CDR2comprising the peptide of SEQ ID NOS:406, 412, 418, 424, 430, 459, 465or 471 or a sequence of amino acids having at least or at least about80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to SEQ ID NOS:406, 412, 418, 424, 430, 459, 465 or 471; a V_(H)CDR3 comprising the peptide of SEQ ID NOS:407, 413, 419, 425, 431, 460,466 or 472 or a sequence of amino acids having at least or at leastabout 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to SEQ ID NOS:407, 413, 419, 425, 431, 460, 466 or472; a V_(L) CDR1 comprising the peptide of SEQ ID NOS:408, 414, 420,426, 432, 461, 467 or 473 or a sequence of amino acids having at leastor at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more sequence identity to SEQ ID NOS:408, 414, 420, 426, 432, 461,467 or 473; a V_(L) CDR2 comprising the peptide of SEQ ID NOS:409, 415,421, 427, 433, 462, 468 or 474 or a sequence of amino acids having atleast or at least about 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NOS:409, 415, 421, 427,433, 462, 468 or 474; and a V_(L) CDR3 comprising the peptide of SEQ IDNOS:410, 416, 422, 428, 434, 463, 469 or 475 or a sequence of aminoacids having at least or at least about 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS:410,416, 422, 428, 434, 463, 469 or 475, wherein the antibody orantigen-binding fragment immunospecifically binds to RSV fusion (F)protein and/or neutralizes RSV.

In particular, provided herein is an antibody containing a VHCDR1 setforth in SEQ ID NO:405 or 437, a VHCDR2 set forth in SEQ ID NO:406, aVHCDR3 set forth in SEQ ID NO:407, a VLCDR1 set forth in SEQ ID NO:408,a VLCDR2 set forth in SEQ ID NO:409, and a VLCDR3 set forth in SEQ IDNO:410, or an antibody containing CDRs that have at least or about 80%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity with any of SEQ ID NOS:405-410 or 437.

Also provided herein is an antibody containing a VHCDR1 set forth in SEQID NO:464 or 483, a VHCDR2 set forth in SEQ ID NO:465, a VHCDR3 setforth in SEQ ID NO:466, a VLCDR1 set forth in SEQ ID NO:467, a VLCDR2set forth in SEQ ID NO:468, and a VLCDR3 set forth in SEQ ID NO:469, oran antibody containing CDRs that have at least or about 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withany of SEQ ID NOS:464-469 or 483.

Also provided herein is an antibody containing a VHCDR1 set forth in SEQID NO:411 or 438, a VHCDR2 set forth in SEQ ID NO:412, a VHCDR3 setforth in SEQ ID NO:413, a VLCDR1 set forth in SEQ ID NO:414, a VLCDR2set forth in SEQ ID NO:415, and a VLCDR3 set forth in SEQ ID NO:416, oran antibody containing CDRs that have at least or about 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withany of SEQ ID NOS:411-416 or 438.

Also provided herein is an antibody containing a VHCDR1 set forth in SEQID NO:417 or 439, a VHCDR2 set forth in SEQ ID NO:418, a VHCDR3 setforth in SEQ ID NO:419, a VLCDR1 set forth in SEQ ID NO:420, a VLCDR2set forth in SEQ ID NO:421, and a VLCDR3 set forth in SEQ ID NO:422, oran antibody containing CDRs that have at least or about 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withany of SEQ ID NOS:417-422 or 439.

Also provided herein is an antibody containing a VHCDR1 set forth in SEQID NO:423 or 440, a VHCDR2 set forth in SEQ ID NO:424, a VHCDR3 setforth in SEQ ID NO:425, a VLCDR1 set forth in SEQ ID NO:426, a VLCDR2set forth in SEQ ID NO:427, and a VLCDR3 set forth in SEQ ID NO:428, oran antibody containing CDRs that have at least or about 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withany of SEQ ID NOS:423-428 or 440.

Also provided herein is an antibody containing a VHCDR1 set forth in SEQID NO:429 or 441, a VHCDR2 set forth in SEQ ID NO:430, a VHCDR3 setforth in SEQ ID NO:431, a VLCDR1 set forth in SEQ ID NO:432, a VLCDR2set forth in SEQ ID NO:433, and a VLCDR3 set forth in SEQ ID NO:434, oran antibody containing CDRs that have at least or about 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withany of SEQ ID NOS:429-434 or 441.

Also provided herein is an antibody containing a VHCDR1 set forth in SEQID NO:458 or 482, a VHCDR2 set forth in SEQ ID NO:459, a VHCDR3 setforth in SEQ ID NO:460, a VLCDR1 set forth in SEQ ID NO:461, a VLCDR2set forth in SEQ ID NO:462, and a VLCDR3 set forth in SEQ ID NO:463, oran antibody containing CDRs that have at least or about 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withany of SEQ ID NOS:458-463 or 482.

Also provided herein is an antibody containing a VHCDR1 set forth in SEQID NO:470 or 484, a VHCDR2 set forth in SEQ ID NO:471, a VHCDR3 setforth in SEQ ID NO:472, a VLCDR1 set forth in SEQ ID NO:473, a VLCDR2set forth in SEQ ID NO:474, and a VLCDR3 set forth in SEQ ID NO:475, oran antibody containing CDRs that have at least or about 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity withany of SEQ ID NOS:470-475 or 484.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a heavy chain, which comprises thepeptide of SEQ ID NO:396. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) domain, whichcomprises the peptide of amino acids 1-121 of SEQ ID NO:396. In someexamples, an isolated antibody or antigen-binding fragment thereofprovided herein contains a light chain, which comprises the peptide ofSEQ ID NO:395. In some examples, an isolated antibody or antigen-bindingfragment provided herein contains a V_(L) domain, which comprises thepeptide of amino acids 1-110 of SEQ ID NO:395. In a particular example,the isolated antibody or antigen-binding fragment thereof is 30D8.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment that contains aheavy chain set forth in SEQ ID NO:396 and a light chain set forth inSEQ ID NO:395.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(H) CDR1, which comprises thepeptide of SEQ ID NO:405 or 437; a V_(H) CDR2, which comprises thepeptide of SEQ ID NO:406; and a V_(H) CDR3, which comprises the peptideof SEQ ID NO:407.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(L) CDR1, which comprises thepeptide of SEQ ID NO:408; a V_(L) CDR2, which comprises the peptide ofSEQ ID NO:409; and a V_(L) CDR3, which comprises the peptide of SEQ IDNO:410.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a heavy chain, which comprises thepeptide of SEQ ID NO:398. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) domain, whichcomprises the peptide of amino acids 1-125 of SEQ ID NO:398. In someexamples, an isolated antibody or antigen-binding fragment thereofprovided herein contains a light chain, which comprises the peptide ofSEQ ID NO:397. In some examples, an isolated antibody or antigen-bindingfragment provided herein contains a V_(L) domain, which comprises thepeptide of amino acids 1-107 of SEQ ID NO:397. In a particular example,the isolated antibody or antigen-binding fragment thereof is 104E5.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment that contains aheavy chain set forth in SEQ ID NO:398 and a light chain set forth inSEQ ID NO:397. In some examples, an isolated antibody or antigen-bindingfragment thereof provided herein contains a V_(H) CDR1, which comprisesthe peptide of SEQ ID NO:411 or 438; a V_(H) CDR2, which comprises thepeptide of SEQ ID NO:412; and a V_(H) CDR3, which comprises the peptideof SEQ ID NO:413.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(L) CDR1, which comprises thepeptide of SEQ ID NO:414; a V_(L) CDR2, which comprises the peptide ofSEQ ID NO:415; and a V_(L) CDR3, which comprises the peptide of SEQ IDNO:416.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a heavy chain, which comprises thepeptide of SEQ ID NO:400. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) domain, whichcomprises the peptide of amino acids 1-124 of SEQ ID NO:400. In someexamples, an isolated antibody or antigen-binding fragment thereofprovided herein contains a light chain, which comprises the peptide ofSEQ ID NO:399. In some examples, an isolated antibody or antigen-bindingfragment provided herein contains a V_(L) domain, which comprises thepeptide of amino acids 1-108 of SEQ ID NO:399. In a particular example,the isolated antibody or antigen-binding fragment thereof is 38F10.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment that contains aheavy chain set forth in SEQ ID NO:400 and a light chain set forth inSEQ ID NO:399.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(H) CDR1, which comprises thepeptide of SEQ ID NO:417 or 439; a V_(H) CDR2, which comprises thepeptide of SEQ ID NO:418; and a V_(H) CDR3, which comprises the peptideof SEQ ID NO:419.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(L) CDR1, which comprises thepeptide of SEQ ID NO:420; a V_(L) CDR2, which comprises the peptide ofSEQ ID NO:421; and a V_(L) CDR3, which comprises the peptide of SEQ IDNO:422.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a heavy chain, which comprises thepeptide of SEQ ID NO:402. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) domain, whichcomprises the peptide of amino acids 1-125 of SEQ ID NO:402. In someexamples, an isolated antibody or antigen-binding fragment thereofprovided herein contains a light chain, which comprises the peptide ofSEQ ID NO:401. In some examples, an isolated antibody or antigen-bindingfragment provided herein contains a V_(L) domain, which comprises thepeptide of amino acids 1-113 of SEQ ID NO:401. In a particular example,the isolated antibody or antigen-binding fragment thereof is 14G3.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment that contains aheavy chain set forth in SEQ ID NO:402 and a light chain set forth inSEQ ID NO:401.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(H) CDR1, which comprises thepeptide of SEQ ID NO:423 or 440; a V_(H) CDR2, which comprises thepeptide of SEQ ID NO:424; and a V_(H) CDR3, which comprises the peptideof SEQ ID NO:425.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(L) CDR1, which comprises thepeptide of SEQ ID NO:426; a V_(L) CDR2, which comprises the peptide ofSEQ ID NO:427; and a V_(L) CDR3, which comprises the peptide of SEQ IDNO:428.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a heavy chain, which comprises thepeptide of SEQ ID NO:404. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) domain, whichcomprises the peptide of amino acids 1-123 of SEQ ID NO:404. In someexamples, an isolated antibody or antigen-binding fragment thereofprovided herein contains a light chain, which comprises the peptide ofSEQ ID NO:403. In some examples, an isolated antibody or antigen-bindingfragment provided herein contains a V_(L) domain, which comprises thepeptide of amino acids 1-107 of SEQ ID NO:403. In a particular example,the isolated antibody or antigen-binding fragment thereof is 90D3.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment that contains aheavy chain set forth in SEQ ID NO:404 and a light chain set forth inSEQ ID NO:403.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(H) CDR1, which comprises thepeptide of SEQ ID NO:429 or 441; a V_(H) CDR2, which comprises thepeptide of SEQ ID NO:430; and a V_(H) CDR3, which comprises the peptideof SEQ ID NO:431.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(L) CDR1, which comprises thepeptide of SEQ ID NO:432; a V_(L) CDR2, which comprises the peptide ofSEQ ID NO:433; and a V_(L) CDR3, which comprises the peptide of SEQ IDNO:434.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a heavy chain, which comprises thepeptide of SEQ ID NO:452. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) domain, whichcomprises the peptide of amino acids 1-124 of SEQ ID NO:452. In someexamples, an isolated antibody or antigen-binding fragment thereofprovided herein contains a light chain, which comprises the peptide ofSEQ ID NO:453. In some examples, an isolated antibody or antigen-bindingfragment provided herein contains a V_(L) domain, which comprises thepeptide of amino acids 1-111 of SEQ ID NO:453. In a particular example,the isolated antibody or antigen-binding fragment thereof is 56E11.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment that contains aheavy chain set forth in SEQ ID NO:452 and a light chain set forth inSEQ ID NO:453.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(H) CDR1, which comprises thepeptide of SEQ ID NO:458 or 482; a V_(H) CDR2, which comprises thepeptide of SEQ ID NO:459; and a V_(H) CDR3, which comprises the peptideof SEQ ID NO:460.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(L) CDR1, which comprises thepeptide of SEQ ID NO:461; a V_(L) CDR2, which comprises the peptide ofSEQ ID NO:462; and a V_(L) CDR3, which comprises the peptide of SEQ IDNO:463.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a heavy chain, which comprises thepeptide of SEQ ID NO:454. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) domain, whichcomprises the peptide of amino acids 1-133 of SEQ ID NO:454. In someexamples, an isolated antibody or antigen-binding fragment thereofprovided herein contains a light chain, which comprises the peptide ofSEQ ID NO:455. In some examples, an isolated antibody or antigen-bindingfragment provided herein contains a V_(L) domain, which comprises thepeptide of amino acids 1-107 of SEQ ID NO:455. In a particular example,the isolated antibody or antigen-binding fragment thereof is 17C9.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment that contains aheavy chain set forth in SEQ ID NO:454 and a light chain set forth inSEQ ID NO:455.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(H) CDR1, which comprises thepeptide of SEQ ID NO:464 or 483; a V_(H) CDR2, which comprises thepeptide of SEQ ID NO:465; and a V_(H) CDR3, which comprises the peptideof SEQ ID NO:466.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(L) CDR1, which comprises thepeptide of SEQ ID NO:467; a V_(L) CDR2, which comprises the peptide ofSEQ ID NO:468; and a V_(L) CDR3, which comprises the peptide of SEQ IDNO:469.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a heavy chain, which comprises thepeptide of SEQ ID NO:456. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) domain, whichcomprises the peptide of amino acids 1-118 of SEQ ID NO:456. In someexamples, an isolated antibody or antigen-binding fragment thereofprovided herein contains a light chain, which comprises the peptide ofSEQ ID NO:457. In some examples, an isolated antibody or antigen-bindingfragment provided herein contains a V_(L) domain, which comprises thepeptide of amino acids 1-109 of SEQ ID NO:457. In a particular example,the isolated antibody or antigen-binding fragment thereof is 69F6.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment that contains aheavy chain set forth in SEQ ID NO:456 and a light chain set forth inSEQ ID NO:457.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(H) CDR1, which comprises thepeptide of SEQ ID NO:470 or 484; a V_(H) CDR2, which comprises thepeptide of SEQ ID NO:471; and a V_(H) CDR3, which comprises the peptideof SEQ ID NO:472.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(L) CDR1, which comprises thepeptide of SEQ ID NO:473; a V_(L) CDR2, which comprises the peptide ofSEQ ID NO:474; and a V_(L) CDR3, which comprises the peptide of SEQ IDNO:475.

Provided herein are isolated anti-RSV antibodies or antigen-bindingfragments thereof that contain a variable heavy (V_(H)) chain and avariable light (V_(L)) chain, where the antibody or antigen-bindingfragment immunospecifically binds to the same epitope on a RSV fusion(F) protein as an antibody or antigen-binding fragment thereof thatcontains a heavy chain set forth in SEQ ID NOS:396, 398, 400, 402, 404,452, 454 or 456 and a light chain set forth in SEQ ID NOS:395, 397, 399,401, 403, 453, 455 or 457.

In some examples, an isolated antibody or antigen-binding fragmentprovided herein contains a V_(H) complementary determining region 1(CDR1), which comprises the peptide of SEQ ID NOS:405, 411, 417, 423,429, 437-441, 458, 464, 470 or 482-484. In some examples, an isolatedantibody or antigen-binding fragment provided herein contains a V_(H)CDR2, which comprises the peptide of SEQ ID NOS:406, 412, 418, 424, 430,459, 465 or 471. In some examples, an isolated antibody orantigen-binding fragment provided herein contains a V_(H) CDR3, whichcomprises the peptide of SEQ ID NOS:407, 413, 419, 425, 431, 460, 466 or472. In some examples, an isolated antibody or antigen-binding fragmentprovided herein contains a V_(L) CDR1, which comprises the peptide ofSEQ ID NOS:408, 414, 420, 426, 432, 461, 467 or 473. In some examples,an isolated antibody or antigen-binding fragment provided hereincontains a V_(L) CDR2, which comprises the peptide of SEQ ID NOS:409,415, 421, 427, 433, 462, 468 or 474. In some examples, an isolatedantibody or antigen-binding fragment provided herein contains a V_(L)CDR3, which comprises the peptide of SEQ ID NO:410, 416, 422, 428, 434,463, 469 or 475.

In some examples, an isolated antibody or antigen-binding fragmentthereof provided herein contains a V_(H) CDR1, wherein the amino acidsequence of the V_(H) CDR1 is set forth in SEQ ID NOS:405, 411, 417,423, 429, 437-441, 458, 464, 470 or 482-484; a V_(H) CDR2, wherein theamino acid sequence of the V_(H) CDR2 is set forth in SEQ ID NOS:406,412, 418, 424, 430, 459, 465 or 471; and a V_(H) CDR3, wherein the aminoacid sequence of the V_(H) CDR3 is set forth in SEQ ID NOS:407, 413,419, 425, 431, 460, 466 or 472. In some examples, an isolated antibodyor antigen-binding fragment thereof provided herein contains a V_(L)CDR1, wherein the amino acid sequence of the V_(L) CDR1 is set forth inSEQ ID NOS:408, 414, 420, 426, 432, 461, 467 or 473; a V_(L) CDR2,wherein the amino acid sequence of the V_(L) CDR2 is set forth in SEQ IDNOS:409, 415, 421, 427, 433, 462, 468 or 474; and a V_(L) CDR3, whereinthe amino acid sequence of the V_(L) CDR3 is set forth in SEQ IDNOS:410, 416, 422, 428, 434, 463, 469 or 475.

Provided herein are isolated polypeptides, antibodies or antigen-bindingfragments thereof that immunospecifically bind to a portion of a RSV Fprotein, which has the amino sequence set forth in SEQ ID NO:25.

Provided herein are isolated polypeptides, antibodies or antigen-bindingfragments thereof that contain an antigen-binding domain that is a humanor a humanized antibody or antigen-binding fragment thereof. In someexamples, the isolated polypeptide, antibody or antigen-binding fragmentprovided herein is a chimeric antibody. In some examples, the isolatedpolypeptide, antibody or antigen-binding fragment is a single-chain Fv(scFv), Fab, Fab′, F(ab′)₂, Fv, dsFv, diabody, Fd, or Fd′ fragment. Insome examples, the isolated polypeptide, antibody or antigen-bindingfragment provided herein contains a peptide linker. In some examples,the peptide linker contains about 1 to about 50 amino acids.

In some examples, the isolated polypeptide, antibody or antigen-bindingfragment thereof provided herein is conjugated to polyethylene glycol(PEG). In some examples, the isolated polypeptide, antibody orantigen-binding fragment provided herein contains a therapeutic ordiagnostic agent. Exemplary diagnostic agents include, but are notlimited to, an enzyme, a fluorescent compound, an electron transferagent, and a radiolabel.

Provided herein are isolated polypeptides, antibodies or antigen-bindingfragments thereof that contain a protein transduction domain. In someexamples, the protein transduction domain is selected from among apeptide having an amino acid sequence set forth in SEQ ID NOS:284-355.In some examples, the protein transduction domain is a HIV-TAT proteintransduction domain.

Provided herein are multivalent antibodies, containing a firstantigen-binding portion containing a polypeptide, antibody orantigen-binding fragment thereof provided herein conjugated to amultimerization domain; and a second antigen-binding portion containingan antigen-binding fragment of an antiviral antibody conjugated to asecond multimerization domain. In such examples, the firstmultimerization domain and the second multimerization domain arecomplementary or the same, whereby the first antigen-binding portion andsecond antigen-binding portion form a multivalent antibody. In someexamples, the multivalent antibodies provided herein contain 1, 2, 3, 4,or 5 additional antigen-binding portions. Exemplary multivalentantibodies include a bivalent, trivalent, tetravalent, pentavalent,hexavalent, or heptavalent antibodies. The multivalent antibodiesprovided herein include heterobivalent or homobivalent antibodies. Themultivalent antibodies provided herein include multispecific antibodies.In some examples, the multispecific antibody is a bispecific,trispecific or tetraspecific antibody. In some examples, the multivalentantibodies provided herein contain an antigen-binding fragment that is asingle-chain Fv (scFv), Fab, Fab′, F(ab′)₂, Fv, dsFv, diabody, Fd, orFd′ fragment. The first antigen-binding portion and/or secondantigen-binding portion of the multivalent antibodies provided hereincan be conjugated to a multimerization domain by covalent ornon-covalent linkage. In some examples, the antigen-binding portion isconjugated to the multimerization domain via a linker, such as achemical linker or a polypeptide linker. In some examples, themultimerization domain of the multivalent antibody provided herein isselected from among an immunoglobulin constant region (Fc), a leucinezipper, complementary hydrophobic regions, complementary hydrophilicregions, or compatible protein-protein interaction domains. In someexamples, the Fc domain is an IgG, IgM or an IgE Fc domain.

In some examples, the multivalent antibodies provided herein contain twoor more anti-RSV antibodies or antigen-binding fragments thereof. In aparticular example, the multivalent antibodies provided herein containtwo or more anti-RSV antibodies or antigen-binding fragments thereof.

Provided herein are multivalent antibodies, containing a firstantigen-binding portion containing an anti-RSV antibody orantigen-binding fragment thereof provided herein conjugated to amultimerization domain; and a second antigen-binding portion containingan anti-RSV antibody or antigen-binding fragment thereof, selected fromamong palivizumab, motavizumab, AFFF, P12f2, P12f4, P11d4, A1e9, A12a6,A13c4, A17d4, A4B4, A8c7, 1X-493L1, FR H3-3F4, M3H9, Y10H6, DG, AFFF(1),6H8, L1-7E5, L2-15B10, A13a11, A1h5, A4B4(1), A4B4L1FR-S28R, A4B4-F52S,rsv6, rsv11, rsv13, rsv19, rsv21, rsv22, rsv23, RF-1, RF-2 or anantigen-binding fragment thereof, conjugated to a second multimerizationdomain.

Provided herein are multivalent antibodies, containing a firstantigen-binding portion containing an anti-RSV antibody orantigen-binding fragment thereof provided herein conjugated to amultimerization domain; and a second antigen-binding portion containingan antiviral antibody that immunospecifically binds an antigen ofparainfluenza virus (PIV) or human metapneumovirus (hMPV), conjugated toa second multimerization domain.

Provided herein are combinations, which contain an isolated polypeptide,antibody or antigen-binding fragment thereof provided herein or amultivalent antibody provided herein, and an antiviral agent. In someexamples, the antiviral agent is ribavirin. Provided herein arecombinations, which contain an isolated polypeptide, antibody orantigen-binding fragment thereof provided herein and an antiviral agentformulated as a single composition or as separate compositions.

Provided herein are combinations, which contain an isolated polypeptide,antibody or antigen-binding fragment thereof provided herein or amultivalent antibody provided herein, and one or more additionalantiviral antibodies. In some examples, the combination contains two ormore different anti-RSV antibodies or antigen-binding fragments thereof.In some examples, the combination contains two or more differentanti-RSV antibodies or antigen-binding fragments selected from among anantibody or antigen-binding fragment provided herein. In some examples,the combination contains an antibody or antigen-binding fragment thereofprovided herein and an antibody selected from among palivizumab,motavizumab, AFFF, P12f2, P12f4, P11d4, A1e9, A12a6, A13c4, A17d4, A4B4,A8c7, 1X-493L1, FR H3-3F4, M3H9, Y10H6, DG, AFFF(1), 6H8, L1-7E5,L2-15B10, A13a11, A1h5, A4B4(1), A4B4L1FR-S28R, A4B4-F52S, rsv6, rsv11,rsv13, rsv19, rsv21, rsv22, rsv23, RF-1, RF-2 or antigen-bindingfragments thereof. In some examples, the combination contains anantibody or antigen-binding fragment thereof provided herein and anantibody selected from among an antibody or antigen-binding fragmentthereof that immunospecifically binds an antigen of parainfluenza virus(PIV) or human metapneumovirus (hMPV). In some examples, the PIV antigenis an antigen of human PIV type 1, human PIV type 2, human PIV type 3,and/or human PIV type 4. In some examples, the PIV antigen is selectedfrom among a PIV nucleocapsid phosphoprotein, a PIV fusion (F) protein,a PIV phosphoprotein, a PIV large (L) protein, a PIV matrix (M) protein,a PIV hemagglutinin-neuraminidase (HN) glycoprotein, a PIV RNA-dependentRNA polymerase, a PIV Y1 protein, a PIV D protein, a PIV C protein, andallelic variants thereof. In some examples, the hMPV antigen is anantigen of hMPV type A or hMPV type B. In some examples, the hMPVantigen is an antigen of hMPV subtype A1, hMPV subtype A2, hMPV subtypeB1, or hMPV subtype B2. In some examples, the hMPV antigen is selectedfrom among a hMPV nucleoprotein, a hMPV phosphoprotein, a hMPV matrixprotein, a hMPV small hydrophobic protein, a hMPV RNA-dependent RNApolymerase, a hMPV F protein, a hMPV G protein, and allelic variantsthereof.

Provided herein are combinations, which contain an isolated polypeptide,antibody or antigen-binding fragment thereof provided herein or amultivalent antibody provided herein, and one or more additionalantiviral antibodies, where the one or more additional antiviralantibodies is a single-chain Fv (scFv), Fab, Fab′, F(ab′)₂, Fv, dsFv,diabody, Fd, or Fd′ fragment.

Provided herein are pharmaceutical compositions containing any isolatedpolypeptide, antibody or antigen-binding fragment thereof providedherein, any multivalent antibody provided herein, or any combinationprovided herein and a pharmaceutically acceptable carrier or excipient.In some examples, the pharmaceutical compositions provided herein areformulated as a gel, ointment, liquid, suspension, aerosol, tablet,pill, powder, or nasal spray. In some examples, the pharmaceuticalcompositions provided herein are formulated for pulmonary, intranasal,or parenteral administration. In some examples, the pharmaceuticalcompositions provided herein are formulated for single dosageadministration. In some examples, the pharmaceutical compositionsprovided herein are formulated for sustained release.

Provided herein are pharmaceutical compositions, which contain anisolated polypeptide, antibody or antigen-binding fragment thereofprovided herein or a multivalent antibody provided herein, and one ormore additional antiviral antibodies. In some examples, thepharmaceutical compositions contain two or more different anti-RSVantibodies or antigen-binding fragments thereof. In some examples, thepharmaceutical compositions contain two or more different anti-RSVantibodies or antigen-binding fragments selected from among an antibodyor antigen-binding fragment provided herein. In some examples, thepharmaceutical compositions contain an antibody or antigen-bindingfragment provided herein and an antibody selected from amongpalivizumab, motavizumab, AFFF, P12f2, P12f4, P11d4, A1e9, A12a6, A13c4,A17d4, A4B4, A8c7, 1X-493L1, FR H3-3F4, M3H9, Y10H6, DG, AFFF(1), 6H8,L1-7E5, L2-15B10, A13a11, A1h5, A4B4(1), A4B4L1FR-S28R, A4B4-F52S, rsv6,rsv11, rsv13, rsv19, rsv21, rsv22, rsv23, RF-1, RF-2 or antigen-bindingfragments thereof. In some examples, the pharmaceutical compositionscontain an antibody or antigen-binding fragment thereof provided hereinand an antibody selected from among an antibody or antigen-bindingfragment thereof that immunospecifically binds an antigen ofparainfluenza virus (PIV) or human metapneumovirus (hMPV). In someexamples, the PIV antigen is an antigen of human PIV type 1, human PIVtype 2, human PIV type 3, and/or human PIV type 4. In some examples, thePly antigen is selected from among a P1V nucleocapsid phosphoprotein, aPIV fusion (F) protein, a PIV phosphoprotein, a PIV large (L) protein, aPIV matrix (M) protein, a PIV hemagglutinin-neuraminidase (HN)glycoprotein, a PIV RNA-dependent RNA polymerase, a PIV Y1 protein, aPIV D protein, a PIV C protein, and allelic variants thereof. In someexamples, the hMPV antigen is an antigen of hMPV type A or hMPV type B.In some examples, the hMPV antigen is an antigen of hMPV subtype A1,hMPV subtype A2, hMPV subtype B1, or hMPV subtype B2. In some examples,the hMPV antigen is selected from among a hMPV nucleoprotein, a hMPVphosphoprotein, a hMPV matrix protein, a hMPV small hydrophobic protein,a hMPV RNA-dependent RNA polymerase, a hMPV F protein, a hMPV G protein,and allelic variants thereof.

Provided herein are pharmaceutical compositions, which contain anisolated polypeptide, antibody or antigen-binding fragment providedherein or a multivalent antibody provided herein, and one or moreadditional antiviral antibodies, where the one or more additionalantiviral antibodies is a single-chain Fv (scFv), Fab, Fab′. F(ab′)₂,Fv, dsFv, diabody, Fd, or Fd′ fragment.

Provided herein are pharmaceutical compositions, which contain anisolated polypeptide, antibody or antigen-binding fragment thereofprovided herein or a multivalent antibody provided herein, and anantiviral agent. In some examples, the antiviral agent is ribavirin.

Provided herein are methods of treating a viral infection in a subject,which involve administering to the subject a therapeutically effectiveamount of a pharmaceutical composition provided herein. Provided hereinare methods of treating or inhibiting one or more symptoms of a viralinfection in a subject, which involve administering to the subject atherapeutically effective amount of a pharmaceutical compositionprovided herein. Also provided herein are methods of preventing a viralinfection in a subject, which involve administering to the subject atherapeutically effective amount of a pharmaceutical compositionprovided herein. In a particular example, the viral infection is a RSVinfection. In a particular example, the RSV infection is an upperrespiratory tract infection.

Administration can be effected by any suitable route, including but notlimited to, topically, parenterally, locally, or systemically, such asfor example intranasally, intramuscularly, intradermally,intraperitoneally, intravenously, subcutaneously, orally, or bypulmonary administration. In some examples, a pharmaceutical compositionprovided herein is administered by a nebulizer or an inhaler. Thepharmaceutical compositions provided herein can be administered to anysuitable subject, such as a mammal, for example, a human.

In some examples, a pharmaceutical composition provided herein isadministered a human infant, a human infant born prematurely or at riskof hospitalization for a RSV infection, an elderly human, a humansubject which has cystic fibrosis, bronchopulmonary dysplasia,congenital heart disease, congenital immunodeficiency, acquiredimmunodeficiency, leukemia, or non-Hodgkin lymphoma or a human subjectwho has had a transplant, such as, for example, a bone marrow transplantor a liver transplant.

In some examples, a pharmaceutical composition provided herein isadministered one time, two times, three times, four times or five timesduring RSV season (e.g., October through May). In some examples, apharmaceutical composition provided herein is administered one time, twotimes, three times, four times or five times within one month, twomonths or three months, prior to a RSV season.

In some examples, a pharmaceutical composition provided herein can beadministered with one or more antiviral agents. In some examples, theantiviral agent is ribavirin. In some examples, the pharmaceuticalcomposition and the antiviral agent are formulated as a singlecomposition or as separate compositions. In the methods provided herein,the pharmaceutical composition and the antiviral agent can beadministered sequentially, simultaneously or intermittently.

In some examples, a pharmaceutical composition provided herein can beadministered with a hormonal therapy, immunotherapy or ananti-inflammatory agent. In some examples, a pharmaceutical compositionprovided herein can be administered with one or more additionalantiviral antibodies or antigen-binding fragments thereof. Thepharmaceutical composition and the one or more additional antiviralantibodies are formulated as a single composition or as separatecompositions. The pharmaceutical composition and the one or moreadditional anti-RSV antibodies can be administered sequentially,simultaneously or intermittently. In some examples, the antigen-bindingfragment is a single-chain Fv (scFv), Fab, Fab′, F(ab′)₂, Fv, dsFv,diabody, Fd, or Fd′ fragment.

In some examples, a pharmaceutical composition provided herein can beadministered with one or more additional antiviral antibodies selectedfrom among anti-RSV antibodies or antigen-binding fragments thereof suchas, for example, palivizumab, motavizumab, AFFF, P12 f2, P12f4, P11d4,A1e9, A12a6, A13c4, A17d4, A4B4, A8c7, 1X-493L1, FR H3-3F4, M3H9, Y10H6,DG, AFFF(1), 6H8, L1-7E5, L2-15B10, A13a11. A1h5, A4B4(l).A4B4L1FR-S28R, A4B4-F52S, rsv6, rsv11, rsv13, rsv19, rsv21, rsv22,rsv23, RF-1, RF-2 or antigen-binding fragments thereof.

In some examples, a pharmaceutical composition provided herein can beadministered with one or more additional antiviral antibodies selectedfrom among an antibody or antigen-binding fragment thereof thatimmunospecifically binds an antigen of parainfluenza virus (PIV) orhuman metapneumovirus (hMPV). In some examples, the PIV antigen is anantigen of human PIV type 1, human PIV type 2, human PIV type 3, and/orhuman PIV type 4. In some examples, the PIV antigen is selected fromamong a PIV nucleocapsid phosphoprotein, a PIV fusion (F) protein, a PIVphosphoprotein, a PIV large (L) protein, a PIV matrix (M) protein, a PIVhemagglutinin-neuraminidase (HN) glycoprotein, a PIV RNA-dependent RNApolymerase, a PIV Y1 protein, a PIV D protein, a PIV C protein, andallelic variants thereof. In some examples, the hMPV antigen is anantigen of hMPV type A or hMPV type B. In some examples, the hMPVantigen is an antigen of hMPV subtype A1, hMPV subtype A2, hMPV subtypeB1, or hMPV subtype B2. In some examples, the hMPV antigen is selectedfrom among a hMPV nucleoprotein, a hMPV phosphoprotein, a hMPV matrixprotein, a hMPV small hydrophobic protein, a hMPV RNA-dependent RNApolymerase, a hMPV F protein, a hMPV G protein, and allelic variantsthereof.

Provided herein are methods of detecting RSV infection, which involve(a) assaying the level of RSV antigen in a fluid, cell, or tissue sampleusing an antibody or antigen-binding fragments thereof provided herein;(b) comparing the assayed level of RSV antigen with a control levelwhereby an increase in the assayed level of RSV antigen compared to thecontrol level of the RSV antigen is indicative of a RSV infection. Insome examples, the cell or tissue sample is obtained from a humansubject. In some examples, the cell or tissue sample is a blood, urine,saliva, lung sputum, lavage, or lymph sample.

Provided herein are isolated nucleic acids that encode the polypeptide,antibody or antigen-binding fragments thereof provided herein. Providedherein are vectors that contain a nucleic acid encoding the polypeptide,antibody or antigen-binding fragments thereof provided herein.

Provided herein are isolated cells that contain an antibody orantigen-binding fragment thereof provided herein, a nucleic acidprovided herein, or a vector provided herein. The cells provided hereincan be, for example, prokaryotic or eukaryotic cells. Also providedherein are transgenic animals that contain a nucleic acid providedherein or a vector provided herein. Also provided herein are methods ofexpressing an isolated antibody or antigen-binding fragment thereof,which involve culturing isolated cells provided herein under conditionswhich express the encoded antibody or by isolation of the antibody orantigen-binding fragment from the transgenic animal provided herein. Insome examples, the antibody or antigen-binding fragment is isolated fromthe serum or milk of the transgenic animal.

Provided herein are kits containing a polypeptide, antibody orantigen-binding fragment of provided herein, a multivalent antibodyprovided herein, or a combination provided herein, in one or morecontainers, and instructions for use.

Also provided herein are uses of an antibody or antigen-binding fragmentthereof provided herein for the prevention of viral infection in asubject. Also provided herein are uses of an antibody or antigen-bindingfragment thereof provided herein for treatment of viral infection in asubject. Also provided herein are uses of an antibody or antigen-bindingfragment thereof provided herein for treating or inhibiting one or moresymptoms of a viral infection in a subject.

Also provided herein are uses of an antibody or antigen-binding fragmentprovided herein for the formulation of a medicament for the preventionof viral infection in a subject. Also provided herein are uses of anantibody or antigen-binding fragment provided herein for the formulationof a medicament for treatment of viral infection in a subject. Alsoprovided herein are uses of an antibody or antigen-binding fragmentprovided herein for the formulation of a medicament for treating orinhibiting one or more symptoms of a viral infection in a subject.

Provided herein are anti-RSV neutralizing antibodies or antigen-bindingfragments thereof, wherein RSV does not produce a virus that escapesneutralization by the antibodies or antigen-binding fragments thereofafter more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morerounds of viral replication in the presence of the antibody orantigen-binding fragment thereof. Also provided herein are anti-RSVneutralizing antibodies or antigen-binding fragments thereof, whereinRSV does not produce a virus that escapes neutralization by theantibodies or antigen-binding fragments thereof after up to 20 rounds ofviral replication in the presence of the antibodies or antigen-bindingfragments thereof. In some examples, the anti-RSV antibody orantigen-binding fragment thereof specifically binds to the same epitopeas the antibody designated 30D8. In other examples, the anti-RSVantibody or antigen-binding fragment thereof is designated 30D8. In someexamples, the anti-RSV antibody or antigen-binding fragment specificallybinds to RSV F protein. In other examples, the anti-RSV antibody orantigen-binding fragment is a Fab or scFv.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a 3D model of the RSV F protein in its prefusion conformation.Amino acid residues identified in Example 12 as being important forbinding of antibody 58C5 are indicated in black, space filling models.When the residues are displayed on the 3D model of prefusion RSV Fprotein, they cluster on a restricted area of the spike and illustratethe antibody footprint of antibody 58C5.

FIG. 2 shows a 3D model X-ray structure of the D3 region of RSV Fprotein containing RSV F protein residues important for binding ofantibody 58C5 in a prefusion and postfusion conformation. As shown inFIG. 2, residues important for binding of antibody 58C5 are clusteredonly in the prefusion model. In the postfusion model, the residues aredistributed over a large area of the protein surface.

FIG. 3 is a 3D model of the RSV F protein in its prefusion conformation.Amino acid residues identified in Example 12 as being important forbinding of antibody 30D8 are indicated in black, space filling models.The residues cluster in the D1 domain at the base of the lollipop justabove the stem/stalk region.

DETAILED DESCRIPTION

Outline

-   A. DEFINITIONS-   B. OVERVIEW

1. Respiratory Syncytial Virus

-   C. ANTI-RSV ANTIBODIES

1. General Antibody Structure and Functional Domains

-   -   a. Structural and Functional Domains of Antibodies    -   b. Antibody Fragments

2. Exemplary Anti-RSV Antibodies

-   -   a. Derivative Antibodies        -   i. Single Chain Antibodies        -   ii. Anti-idiotypic Antibodies        -   iii. Multi-specific Antibodies and Antibody Multimerization

-   D. ADDITIONAL MODIFICATIONS OF ANTI-RSV ANTIBODIES

1. Modifications to reduce immunogenicity

2. Fc Modifications

3. Pegylation

4. Conjugation of a Detectable Moiety

5. Conjugation of a Therapeutic Moiety

6. Modifications to improve binding specificity

-   E. METHODS OF ISOLATING ANTI-RSV ANTIBODIES-   F. METHODS OF PRODUCING ANTI-RSV ANTIBODIES, AND MODIFIED OR VARIANT    FORMS THEREOF AND NUCLEIC ACIDS ENCODING ANTIBODIES

1. Nucleic Acids

2. Vectors

3. Cell Expression Systems

-   -   a. Prokaryotic Expression    -   b. Yeast Cells    -   c. Insect Cells    -   d. Mammalian Cells    -   e. Plants

4. Purification of Antibodies

-   G. ASSESSING ANTI-RSV ANTIBODY PROPERTIES AND ACTIVITIES

1. Binding Assays

2. Binding specificity

3. In vitro assays for analyzing virus neutralization effects ofantibodies

4. In vivo animal models for assessing efficacy of the anti-RSVantibodies

5. In vitro and in vivo Assays for Measuring Antibody Efficacy

-   H. DIAGNOSTIC USES

1. In vitro detection of pathogenic infection

2. In vivo detection of pathogenic infection

3. Monitoring Infection

-   I. PROPHYLACTIC AND THERAPEUTIC USES

1. Subjects for therapy

2. Dosages

3. Routes of Administration

4. Combination therapies

-   -   a. Antiviral Antibodies for Combination Therapy        -   i. Anti-RSV antibodies        -   ii. Antibodies against other respiratory viruses

5. Gene Therapy

-   J. Pharmaceutical Compositions, Combinations and Articles of    manufacture/Kits

1. Pharmaceutical Compositions

2. Articles of Manufacture/Kits

3. Combinations

-   K. Examples

Example 1: Expression of RSV F protein

Example 2: Isolation of Anti-RSV Fab Antibodies from EBV-transformed BCells

Example 3: Isolation of Anti-RSV Fab Antibodies by Single Cell Sorting

Example 4: Antibody Binding to RSV F protein

Example 5: Expression and Purification of Isolated Fabs

Example 6: RSV Neutralization Assay

Example 7: Cloning and Expression of IgG

Example 8: IgG Binding Assays

Example 9: IgG RSV Neutralization Assays

Example 10: Competition Assays

Example 11: RSV MARM Generation and Neutralization Assays

Example 12: Mapping of the 58C5 and 30D8 Binding Epitopes

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, GenBank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, itunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, “antibody” refers to immunoglobulins and immunoglobulinfragments, whether natural or partially or wholly synthetically, such asrecombinantly, produced, including any fragment thereof containing atleast a portion of the variable region of the immunoglobulin moleculethat retains the binding specificity ability of the full-lengthimmunoglobulin. Hence, an antibody includes any protein having a bindingdomain that is homologous or substantially homologous to animmunoglobulin antigen-binding domain (antibody combining site).Antibodies include antibody fragments, such as anti-RSV antibodyfragments. As used herein, the term antibody, thus, includes syntheticantibodies, recombinantly produced antibodies, multispecific antibodies(e.g., bispecific antibodies), human antibodies, non-human antibodies,humanized antibodies, chimeric antibodies, intrabodies, and antibodyfragments, such as, but not limited to, Fab fragments, Fab′ fragments,F(ab′)₂ fragments, Fv fragments, disulfide-linked Fvs (dsFv), Fdfragments, Fd′ fragments, single-chain Fvs (scFv), single-chain Fabs(scFab), diabodies, anti-idiotypic (anti-Id) antibodies, orantigen-binding fragments of any of the above. Antibodies providedherein include members of any immunoglobulin type (e.g., IgG, IgM, IgD,IgE, IgA and IgY), any class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 andIgA2) or subclass (e.g., IgG2a and IgG2b).

As used herein, an “antibody fragment” or “antigen-binding fragment” ofan antibody refers to any portion of a full-length antibody that is lessthan full length but contains at least a portion of the variable regionof the antibody that binds antigen (e.g. one or more CDRs and/or one ormore antibody combining sites) and thus retains the binding specificity,and at least a portion of the specific binding ability of thefull-length antibody. Hence, an antigen-binding fragment refers to anantibody fragment that contains an antigen-binding portion that binds tothe same antigen as the antibody from which the antibody fragment isderived. Antibody fragments include antibody derivatives produced byenzymatic treatment of full-length antibodies, as well as synthetically,e.g. recombinantly produced derivatives. An antibody fragment isincluded among antibodies. Examples of antibody fragments include, butare not limited to, Fab, Fab′, F(ab′)₂, single-chain Fv (scFv), Fv,dsFv, diabody, Fd and Fd′ fragments and other fragments, includingmodified fragments (see, for example, Methods in Molecular Biology, Vol207: Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003); Chapter 1; p 3-25, Kipriyanov). The fragment can includemultiple chains linked together, such as by disulfide bridges and/or bypeptide linkers. An antibody fragment generally contains at least orabout 50 amino acids and typically at least or about 200 amino acids. Anantigen-binding fragment includes any antibody fragment that wheninserted into an antibody framework (such as by replacing acorresponding region) results in an antibody that immunospecificallybinds (i.e. exhibits Ka of at least or at least about 10⁷-10⁸ M⁻¹) tothe antigen.

As used herein, a “therapeutic antibody” refers to any antibody orantigen-binding fragment thereof that is administered for treatment ofan animal, including a human. Such antibodies can be prepared by anyknown methods for the production of polypeptides, and hence, include,but are not limited to, recombinantly produced antibodies, syntheticallyproduced antibodies, and therapeutic antibodies extracted from cells ortissues and other sources. As isolated from any sources or as produced,therapeutic antibodies can be heterogeneous in length or differ inpost-translational modification, such as glycosylation (i.e.carbohydrate content). Heterogeneity of therapeutic antibodies also candiffer depending on the source of the therapeutic antibodies. Hence,reference to therapeutic antibodies refers to the heterogeneouspopulation as produced or isolated. When a homogeneous preparation isintended, it will be so-stated. References to therapeutic antibodiesherein are to their monomeric, dimeric or other multimeric forms, asappropriate.

As used herein, a “neutralizing antibody” is any antibody orantigen-binding fragment thereof that binds to a pathogen and interfereswith the ability of the pathogen to infect a cell and/or cause diseasein a subject. Exemplary of neutralizing antibodies are neutralizingantibodies that bind to viruses, bacteria, and fungal pathogens.Typically, the neutralizing antibodies provide herein bind to thesurface of the pathogen. In examples where the pathogen is a virus, aneutralizing antibody that binds to the virus typically hinds to aprotein on the surface of the virus. Depending on the class of thevirus, the surface protein can be a capsid protein (e.g. a capsidprotein of a non-enveloped virus) or a viral envelope protein (e.g., aviral envelope protein of an enveloped virus). In some examples, theprotein is a glycoprotein. The ability of the virus to inhibit virusinfectivity can be measure for example, by an in vitro neutralizationassay, such as, for example, a plaque reduction assay using Vero hostcells.

As used herein, an “enveloped virus” is an animal virus which possessesan outer membrane or envelope, which is a lipid bilayer containing viralproteins, surrounding the virus capsid. The envelope proteins of thevirus participate in the assembly of the infectious particle and alsoare involved in virus entry by binding to receptors present on the hostcell and inducing fusion between the viral envelope and a membrane ofthe host cell. Enveloped viruses can be either spherical or filamentous(rod-shaped). Exemplary enveloped viruses include, but are not limitedto, members of the Herpesviridae, Poxviridae, Hepadnaviridae,Togaviridae, Arenaviridae, Flaviviridae, Orthomyxoviridae,Paramyxoviridae, Bunyaviridae, Rhabdoviridae, Filoviridae,Coronaviridae, and Bornaviridae virus families. RSV is a negative sensesingle stranded RNA enveloped virus of the Paramyxoviridae family,Pneumovirinae subfamily.

As used herein, a “non-enveloped virus” or “naked virus” is a virus thatlacks a viral envelope. For infection of a host cell, a non-envelopedvirus uses proteins of the viral capsid for attachment to the targetcell. Exemplary non-enveloped viruses include, but are not limited to,Adenoviridae, Papillomavirinae, Parvoviridae, Polyomavirinae,Circoviridae, Reoviridae, Picornaviridae, Caliciviridae, andAstroviridae virus families.

As used herein, a “surface protein” of a pathogen is any protein that islocated on external surface of the pathogen. The surface protein can bepartially or entirely exposed to the external environment (i.e. outersurface). Exemplary of surface proteins are membrane proteins, such as,for example, a protein located on the surface of a viral envelope orbacterial outer membrane (e.g., a membrane glycoprotein). Membraneproteins can be transmembrane proteins (i.e. proteins that traverse thelipid bilayer) or proteins that are non-transmembrane cell surfaceassociated proteins (e.g., anchored or covalently attached to thesurface of the membrane, such as attachment to another protein on thesurface of the pathogen). Other exemplary surface proteins include viralcapsid proteins of non-enveloped enveloped viruses that are at leastpartially exposed to the external environment.

As used herein, “monoclonal antibody” refers to a population ofidentical antibodies, meaning that each individual antibody molecule ina population of monoclonal antibodies is identical to the others. Thisproperty is in contrast to that of a polyclonal population ofantibodies, which contains antibodies having a plurality of differentsequences. Monoclonal antibodies can be produced by a number ofwell-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917;and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example,monoclonal antibodies can be produced by immortalization of a B cell,for example through fusion with a myeloma cell to generate a hybridomacell line or by infection of B cells with virus such as EBV. Recombinanttechnology also can be used to produce antibodies in vitro from clonalpopulations of host cells by transforming the host cells with plasmidscarrying artificial sequences of nucleotides encoding the antibodies.

As used herein, a “conventional antibody” refers to an antibody thatcontains two heavy chains (which can be denoted H and H′) and two lightchains (which can be denoted L and L′) and two antibody combining sites,where each heavy chain can be a full-length immunoglobulin heavy chainor any functional region thereof that retains antigen-binding capability(e.g. heavy chains include, but are not limited to, V_(H), chainsV_(H)-C_(H)1 chains and V_(H)-C_(H)1-C_(H)2-C_(H)3 chains), and eachlight chain can be a full-length light chain or any functional region of(e.g. light chains include, but are not limited to, V_(L) chains andV_(L)-C_(L) chains). Each heavy chain (H and H′) pairs with one lightchain (L and L′, respectively)

As used herein, a full-length antibody is an antibody having twofull-length heavy chains (e.g. V_(H)-C_(H)1-C_(H)2-C_(H)3 orV_(H)-C_(H)1-C_(H)2-C_(H)3-C_(H)4) and two full-length light chains(V_(L)-C_(L)) and hinge regions, such as human antibodies producednaturally by antibody secreting B cells and antibodies with the samedomains that are synthetically produced.

As used herein, an Fv antibody fragment is composed of one variableheavy domain (V_(H)) and one variable light (V_(L)) domain linked bynoncovalent interactions.

As used herein, a dsFv refers to an Fv with an engineered intermoleculardisulfide bond, which stabilizes the V_(H)-V_(L) pair.

As used herein, an Fd fragment is a fragment of an antibody containing avariable domain (V_(H)) and one constant region domain (C_(H)1) of anantibody heavy chain.

As used herein, a Fab fragment is an antibody fragment that results fromdigestion of a full-length immunoglobulin with papain, or a fragmenthaving the same structure that is produced synthetically, e.g. byrecombinant methods. A Fab fragment contains a light chain (containing aV_(L) and C_(L)) and another chain containing a variable domain of aheavy chain (V_(H)) and one constant region domain of the heavy chain(C_(H)1).

As used herein, a F(ab′)₂ fragment is an antibody fragment that resultsfrom digestion of an immunoglobulin with pepsin at pH 4.0-4.5, or afragment having the same structure that is produced synthetically, e.g.by recombinant methods. The F(ab′)₂ fragment essentially contains twoFab fragments where each heavy chain portion contains an additional fewamino acids, including cysteine residues that form disulfide linkagesjoining the two fragments.

As used herein, a Fab′ fragment is a fragment containing one half (oneheavy chain and one light chain) of the F(ab′)₂ fragment.

As used herein, an Fd′ fragment is a fragment of an antibody containingone heavy chain portion of a F(ab′)₂ fragment.

As used herein, an Fv′ fragment is a fragment containing only the V_(H)and V_(L) domains of an antibody molecule.

As used herein, hsFv refers to antibody fragments in which the constantdomains normally present in a Fab fragment have been substituted with aheterodimeric coiled-coil domain (see, e.g., Arndt et al. (2001) J MolBiol. 7:312:221-228).

As used herein, an scFv fragment refers to an antibody fragment thatcontains a variable light chain (V_(L)) and variable heavy chain(V_(H)), covalently connected by a polypeptide linker in any order. Thelinker is of a length such that the two variable domains are bridgedwithout substantial interference. Exemplary linkers are (Gly-Ser)_(n)residues with some Glu or Lys residues dispersed throughout to increasesolubility.

As used herein, the term “derivative” refers to a polypeptide thatcontains an amino acid sequence of an anti-RSV antibody or a fragmentthereof which has been modified, for example, by the introduction ofamino acid residue substitutions, deletions or additions, by thecovalent attachment of any type of molecule to the polypeptide (e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein). A derivativeof an anti-RSV antibody or antigen-binding fragment thereof can bemodified by chemical modifications using techniques known to those ofskill in the art, including, but not limited to, specific chemicalcleavage, acetylation, formylation. Further, a derivative of an anti-RSVantibody or antigen-binding fragment thereof can contain one or morenon-classical amino acids. Typically, a polypeptide derivative possessesa similar or identical function as an anti-RSV antibody orantigen-binding fragment thereof provided herein (e.g., neutralizationof RSV).

As used herein, the phrase “derived from” when referring to antibodyfragments derived from another antibody, such as a monoclonal antibody,refers to the engineering of antibody fragments (e.g., Fab, F(ab′),F(ab′)₂, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd′fragments) that retain the binding specificity of the original antibody.Such fragments can be derived by a variety of methods known in the art,including, but not limited to, enzymatic cleavage, chemicalcrosslinking, recombinant means or combinations thereof. Generally, thederived antibody fragment shares the identical or substantiallyidentical heavy chain variable region (V_(H)) and light chain variableregion (V_(L)) of the parent antibody, such that the antibody fragmentand the parent antibody bind the same epitope.

As used herein, a “parent antibody” or “source antibody” refers the toan antibody from which an antibody fragment (e.g., Fab, F(ab′), F(ab′)₂,single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments) isderived.

As used herein, the term “epitope” refers to any antigenic determinanton an antigen to which the paratope of an antibody binds. Epitopicdeterminants typically contain chemically active surface groupings ofmolecules such as amino acids or sugar side chains and typically havespecific three dimensional structural characteristics, as well asspecific charge characteristics.

As used herein, a chimeric polypeptide refers to a polypeptide thatcontains portions from at least two different polypeptides or from twonon-contiguous portions of a single polypeptide. Thus, a chimericpolypeptide generally includes a sequence of amino acid residues fromall or part of one polypeptide and a sequence of amino acids from all orpart of another different polypeptide. The two portions can be linkeddirectly or indirectly and can be linked via peptide bonds, othercovalent bonds or other non-covalent interactions of sufficient strengthto maintain the integrity of a substantial portion of the chimericpolypeptide under equilibrium conditions and physiologic conditions,such as in isotonic pH 7 buffered saline. For purposes herein, chimericpolypeptides include those containing all or part of an anti-RSVantibody linked to another polypeptide, such as, for example, amultimerization domain, a heterologous immunoglobulin constant domain orframework region, or a diagnostic or therapeutic polypeptide.

As used herein, a fusion protein is a polypeptide engineered to containsequences of amino acids corresponding to two distinct polypeptides,which are joined together, such as by expressing the fusion protein froma vector containing two nucleic acids, encoding the two polypeptides, inclose proximity, e.g., adjacent, to one another along the length of thevector. Generally, a fusion protein provided herein refers to apolypeptide that contains a polypeptide having the amino acid sequenceof an antibody or antigen-binding fragment thereof and a polypeptide orpeptide having the amino acid sequence of a heterologous polypeptide orpeptide, such as, for example, a diagnostic or therapeutic polypeptide.Accordingly, a fusion protein refers to a chimeric protein containingtwo, or portions from two, or more proteins or peptides that are linkeddirectly or indirectly via peptide bonds. The two molecules can beadjacent in the construct or separated by a linker, or spacerpolypeptide. The spacer can encode a polypeptide that alters theproperties of the polypeptide, such as solubility or intracellulartrafficking.

As used herein, “linker” or “spacer” peptide refers to short sequencesof amino acids that join two polypeptide sequences (or nucleic acidencoding such an amino acid sequence). “Peptide linker” refers to theshort sequence of amino acids joining the two polypeptide sequences.Exemplary of polypeptide linkers are linkers joining a peptidetransduction domain to an antibody or linkers joining two antibodychains in a synthetic antibody fragment such as an scFv fragment.Linkers are well-known and any known linkers can be used in the providedmethods. Exemplary of polypeptide linkers are (Gly-Ser)_(n) amino acidsequences, with some Glu or Lys residues dispersed throughout toincrease solubility. Other exemplary linkers are described herein; anyof these and other known linkers can be used with the providedcompositions and methods.

As used herein, “antibody hinge region” or “hinge region” refers to apolypeptide region that exists naturally in the heavy chain of thegamma, delta and alpha antibody isotypes, between the C_(H)1 and C_(H)2domains that has no homology with the other antibody domains. Thisregion is rich in proline residues and gives the IgG, IgD and IgAantibodies flexibility, allowing the two “arms” (each containing oneantibody combining site) of the Fab portion to be mobile, assumingvarious angles with respect to one another as they bind antigen. Thisflexibility allows the Fab arms to move in order to align the antibodycombining sites to interact with epitopes on cell surfaces or otherantigens. Two interchain disulfide bonds within the hinge regionstabilize the interaction between the two heavy chains. In someembodiments provided herein, the synthetically produced antibodyfragments contain one or more hinge regions, for example, to promotestability via interactions between two antibody chains. Hinge regionsare exemplary of dimerization domains.

As used herein, diabodies are dimeric scFv; diabodies typically haveshorter peptide linkers than scFvs, and preferentially dimerize.

As used herein, humanized antibodies refer to antibodies that aremodified to include “human” sequences of amino acids so thatadministration to a human does not provoke an immune response. Ahumanized antibody typically contains complementarily determiningregions (CDRs) derived from a non-human species immunoglobulin and theremainder of the antibody molecule derived mainly from a humanimmunoglobulin. Methods for preparation of such antibodies are known.For example, DNA encoding a monoclonal antibody can be altered byrecombinant DNA techniques to encode an antibody in which the amino acidcomposition of the non-variable regions is based on human antibodies.Methods for identifying such regions are known, including computerprograms, which are designed for identifying the variable andnon-variable regions of immunoglobulins.

As used herein, idiotype refers to a set of one or more antigenicdeterminants specific to the variable region of an immunoglobulinmolecule.

As used herein, anti-idiotype antibody refers to an antibody directedagainst the antigen-specific part of the sequence of an antibody or Tcell receptor. In principle an anti-idiotype antibody inhibits aspecific immune response.

As used herein, an Ig domain is a domain, recognized as such by those inthe art, that is distinguished by a structure, called the Immunoglobulin(Ig) fold, which contains two beta-pleated sheets, each containinganti-parallel beta strands of amino acids connected by loops. The twobeta sheets in the Ig fold are sandwiched together by hydrophobicinteractions and a conserved intra-chain disulfide bond. Individualimmunoglobulin domains within an antibody chain further can bedistinguished based on function. For example, a light chain contains onevariable region domain (V_(L)) and one constant region domain (C_(L)),while a heavy chain contains one variable region domain (V_(H)) andthree or four constant region domains (C_(H)). Each V_(L), C_(L), V_(H),and C_(H) domain is an example of an immunoglobulin domain.

As used herein, a variable domain or variable region is a specific Igdomain of an antibody heavy or light chain that contains a sequence ofamino acids that varies among different antibodies. Each light chain andeach heavy chain has one variable region domain. V_(L) and V_(H),respectively. The variable domains provide antigen specificity, and thusare responsible for antigen recognition. Each variable region containsCDRs that are part of the antigen-binding site domain and frameworkregions (FRs).

As used herein, “antigen-binding domain,” “antigen-binding site,”“antigen combining site” and “antibody combining site” are usedsynonymously to refer to a domain within an antibody that recognizes andphysically interacts with cognate antigen. A native conventionalfull-length antibody molecule has two conventional antigen-bindingsites, each containing portions of a heavy chain variable region andportions of a light chain variable region. A conventionalantigen-binding site contains the loops that connect the anti-parallelbeta strands within the variable region domains. The antigen combiningsites can contain other portions of the variable region domains. Eachconventional antigen-binding site contains three hypervariable regionsfrom the heavy chain and three hypervariable regions from the lightchain. The hypervariable regions also are calledcomplementarity-determining regions (CDRs).

As used herein, “hypervariable region,” “HV,”“complementarity-determining region” and “CDR” and “antibody CDR” areused interchangeably to refer to one of a plurality of portions withineach variable region that together form an antigen-binding site of anantibody. Each variable region domain contains three CDRs, named CDR1,CDR2 and CDR3. The three CDRs are non-contiguous along the linear aminoacid sequence, but are proximate in the folded polypeptide. The CDRs arelocated within the loops that join the parallel strands of the betasheets of the variable domain. As described herein, one of skill in theart knows and can identify the CDRs based on Kabat or Chothia numbering(see e.g., Kabat, E. A. et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, and Chothia, C. et al.(1987) J. Mol. Biol. 196:901-917).

As used herein, framework regions (FRs) are the domains within theantibody variable region domains that are located within the betasheets; the FR regions are comparatively more conserved, in terms oftheir amino acid sequences, than the hypervariable regions.

As used herein, a “constant region” domain is a domain in an antibodyheavy or light chain that contains a sequence of amino acids that iscomparatively more conserved than that of the variable region domain. Inconventional full-length antibody molecules, each light chain has asingle light chain constant region (C_(L)) domain and each heavy chaincontains one or more heavy chain constant region (C_(H)) domains, whichinclude, C_(H)1, C_(H)2, C_(H)3 and C_(H)4. Full-length IgA, IgD and IgGisotypes contain C_(H)1, C_(H)2, C_(H)3 and a hinge region, while IgEand IgM contain C_(H)1, C_(H)2, C_(H)3 and C_(H)4, C_(H)1 and C_(L)domains extend the Fab arm of the antibody molecule, thus contributingto the interaction with antigen and rotation of the antibody arms.Antibody constant regions can serve effector functions, such as, but notlimited to, clearance of antigens, pathogens and toxins to which theantibody specifically binds, e.g., through interactions with variouscells, biomolecules and tissues.

As used herein, a functional region of an antibody is a portion of theantibody that contains at least a V_(H), V_(L), C_(H) (e.g. C_(H)1,C_(H)2 or C_(H)3). C_(L) or hinge region domain of the antibody, or atleast a functional region thereof.

As used herein, a functional region of a V_(H) domain is at least aportion of the full V_(H) domain that retains at least a portion of thebinding specificity of the full V_(H) domain (e.g. by retaining one ormore CDR of the full V_(H) domain), such that the functional region ofthe V_(H) domain, either alone or in combination with another antibodydomain (e.g. V_(L) domain) or region thereof binds to antigen. Exemplaryfunctional regions of V_(H) domains are regions containing the CDR1,CDR2 and/or CDR3 of the V_(H) domain.

As used herein, a functional region of a V_(L) domain is at least aportion of the full V_(L) domain that retains at least a portion of thebinding specificity of the full V_(L) domain (e.g. by retaining one ormore CDRs of the full V_(L) domain), such that the function region ofthe V_(L) domain, either alone or in combination with another antibodydomain (e.g. V_(H) domain) or region thereof, binds to antigen.Exemplary functional regions of V_(L) domains are regions containing theCDR1, CDR2 and/or CDR3 of the V_(L) domain.

As used herein, “specifically bind” or “immunospecifically bind” withrespect to an antibody or antigen-binding fragment thereof are usedinterchangeably herein and refer to the ability of the antibody orantigen-binding fragment to form one or more noncovalent bonds with acognate antigen, by noncovalent interactions between the antibodycombining site(s) of the antibody and the antigen. The antigen can be anisolated antigen or presented in a virus. Typically, an antibody thatimmunospecifically binds (or that specifically binds) to a virus antigenor virus is one that binds to the virus antigen (or to the antigen inthe virus or to the virus) with an affinity constant Ka of about or1×10⁷ M⁻¹ or 1×10⁸ M⁻¹ or greater (or a dissociation constant (K_(d)) of1×10⁻⁷ M or 1×10⁻⁸ M or less). Affinity constants can be determined bystandard kinetic methodology for antibody reactions, for example,immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000)Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599),isothermal titration calorimetry (ITC) or other kinetic interactionassays known in the art (see, e.g., Paul, ed., Fundamental Immunology,2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Pat.No. 7,229,619 for a description of exemplary SPR and ITC methods forcalculating the binding affinity of anti-RSV antibodies).Instrumentation and methods for real time detection and monitoring ofbinding rates are known and are commercially available (e.g., BiaCore2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences;Malmqvist (2000) Biochem. Soc. Trans. 27:335). An antibody thatimmunospecifically binds to a virus antigen (or virus) can bind to otherpeptides, polypeptides, or proteins or viruses with equal or lowerbinding affinity. Typically, an antibody or antigen-binding fragmentthereof provided herein that binds immunospecifically to a RSV F protein(or RSV virus) does not cross-react with other antigens or cross reactswith substantially (at least 10-100 fold) lower affinity for suchantigens. Antibodies or antigen-binding fragments thatimmunospecifically bind to a particular virus antigen (e.g. a RSV Fprotein) can be identified, for example, by immunoassays, such asradioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISAs),surface plasmon resonance, or other techniques known to those of skillin the art. An antibody or antigen-binding fragment thereof thatimmunospecifically binds to an epitope on a RSV F protein typically isone that binds to the epitope (presented in the protein or virus) with ahigher binding affinity than to any cross-reactive epitope as determinedusing experimental techniques, such as, but not limited to,immunoassays, surface plasmon resonance, or other techniques known tothose of skill in the art. Immunospecific binding to an isolated RSVprotein (i.e., a recombinantly produced protein), such as RSV F protein,does not necessarily mean that the antibody will exhibit the sameimmunospecific binding and/or neutralization of the virus. Suchmeasurements and properties are distinct. The affinity for the antibodyor antigen-binding fragments for virus or the antigen as presented inthe virus can be determined. For purposes herein, when describing anaffinity or related term, the target, such as the isolated protein orthe virus, will be identified.

As used herein, the term “surface plasmon resonance” refers to anoptical phenomenon that allows for the analysis of real-timeinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example, using the BiaCore system (GEHealthcare Life Sciences).

As used herein, a “multivalent” antibody is an antibody containing twoor more antigen-binding sites. Multivalent antibodies encompassbivalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalentor higher valency antibodies.

As used herein, a “monospecific” is an antibody that contains two ormore antigen-binding sites, where each antigen-binding siteimmunospecifically binds to the same epitope.

As used herein, a “multispecific” antibody is an antibody that containstwo or more antigen-binding sites, where at least two of theantigen-binding sites immunospecifically bind to different epitopes.

As used herein, a “bispecific” antibody is a multispecific antibody thatcontains two or more antigen-binding sites and can immunospecificallybind to two different epitopes. A “trispecific” antibody is amultispecific antibody that contains three or more antigen-binding sitesand can immunospecifically bind to three different epitopes, a“tetraspecific” antibody is a multispecific antibody that contains fouror more antigen-binding sites and can immunospecifically bind to fourdifferent epitopes, and so on.

As used herein, a “heterobivalent” antibody is a bispecific antibodythat contains two antigen-binding sites, where each antigen-binding siteimmunospecifically binds to a different epitope.

As used herein, a “homobivalent” antibody is a monospecific antibodythat contains two antigen-binding sites, where each antigen-binding siteimmunospecifically binds to the same epitope. Homobivalent antibodiesinclude, but are not limited to, conventional full length antibodies,engineered or synthetic full-length antibodies, any multimer of twoidentical antigen-binding fragments, or any multimer two antigen-bindingfragments containing the same antigen-binding domain.

As used herein, a multimerization domain refers to a sequence of aminoacids that promotes stable interaction of a polypeptide molecule withone or more additional polypeptide molecules, each containing acomplementary multimerization domain, which can be the same or adifferent multimerization domain to form a stable multimer with thefirst domain. Generally, a polypeptide is joined directly or indirectlyto the multimerization domain. Exemplary multimerization domains includethe immunoglobulin sequences or portions thereof leucine zippers,hydrophobic regions, hydrophilic regions, and compatible protein-proteininteraction domains. The multimerization domain, for example, can be animmunoglobulin constant region or domain, such as, for example, the Fcdomain or portions thereof from IgG, including IgG1, IgG2, IgG3 or IgG4subtypes, IgA, IgE, IgD and IgM and modified forms thereof.

As used herein, dimerization domains are multimerization domains thatfacilitate interaction between two polypeptide sequences (such as, butnot limited to, antibody chains). Dimerization domains include, but arenot limited to, an amino acid sequence containing a cysteine residuethat facilitates formation of a disulfide bond between two polypeptidesequences, such as all or part of a full-length antibody hinge region,or one or more dimerization sequences, which are sequences of aminoacids known to promote interaction between polypeptides (e.g., leucinezippers. GCN4 zippers).

As used herein, “Fe” or “Fc region” or “Fc domain” refers to apolypeptide containing the constant region of an antibody heavy chain,excluding the first constant region immunoglobulin domain. Thus, Fcrefers to the last two constant region immunoglobulin domains of IgA,IgD, and IgE, or the last three constant region immunoglobulin domainsof IgE and IgM. Optionally, an Fe domain can include all or part of theflexible hinge N-terminal to these domains. For IgA and IgM. Fc caninclude the 0.1 chain. For an exemplary Fc domain of IgG, Fc containsimmunoglobulin domains Cγ2 and Cγ3, and optionally, all or part of thehinge between Cγ1 and Cγ2. The boundaries of the Fc region can vary, buttypically, include at least part of the hinge region. In addition, Fcalso includes any allelic or species variant or any variant or modifiedform, such as any variant or modified form that alters the binding to anFcR or alters an Fe-mediated effector function.

As used herein, “Fc chimera” refers to a chimeric polypeptide in whichone or more polypeptides is linked, directly or indirectly, to an Fcregion or a derivative thereof. Typically, an Fc chimera combines the Fcregion of an immunoglobulin with another polypeptide, such as forexample an anti-RSV antibody fragment. Derivatives of or modified Fcpolypeptides are known to those of skill in the art.

As used herein, a “protein transduction domain” or “PTD” is a peptidedomain that can be conjugated to a protein, such as an antibody providedherein, to promote the attachment to and/or uptake of the protein into atarget cell.

As used herein, a “tag” or an “epitope tag” refers to a sequence ofamino acids, typically added to the N- or C-terminus of a polypeptide,such as an antibody provided herein. The inclusion of tags fused to apolypeptide can facilitate polypeptide purification and/or detection.Typically, a tag or tag polypeptide refers to polypeptide that hasenough residues to provide an epitope recognized by an antibody or canserve for detection or purification, yet is short enough such that itdoes not interfere with activity of chimeric polypeptide to which it islinked. The tag polypeptide typically is sufficiently unique so anantibody that specifically binds thereto does not substantiallycross-react with epitopes in the polypeptide to which it is linked.Suitable tag polypeptides generally have at least 5 or 6 amino acidresidues and usually between about 8-50 amino acid residues, typicallybetween 9-30 residues. The tags can be linked to one or more chimericpolypeptides in a multimer and permit detection of the multimer or itsrecovery from a sample or mixture. Such tags are well known and can bereadily synthesized and designed. Exemplary tag polypeptides includethose used for affinity purification and include, His tags, theinfluenza hemagglutinin (HA) tag polypeptide and its antibody 12CA5,(Field et al. (1988) Mol. Cell. Biol. 8:2159-2165); the c-myc tag andthe 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (see, e.g., Evanet al. (1985) Molecular and Cellular Biology 5:3610-3616); and theHerpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborskyet al. (1990) Protein Engineering 3:547-553 (1990). An antibody used todetect an epitope-tagged antibody is typically referred to herein as asecondary antibody.

As used herein, “polypeptide” refers to two or more amino acidscovalently joined. The terms “polypeptide” and “protein” are usedinterchangeably herein.

As used herein, a “peptide” refers to a polypeptide that is from 2 toabout or 40 amino acids in length.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids contained in theantibodies provided include the twenty naturally-occurring amino acids(Table 1), non-natural amino acids, and amino acid analogs (e.g., aminoacids wherein the α-carbon has a side chain). As used herein, the aminoacids, which occur in the various amino acid sequences of polypeptidesappearing herein, are identified according to their well-known,three-letter or one-letter abbreviations (see Table 1). The nucleotides,which occur in the various nucleic acid molecules and fragments, aredesignated with the standard single-letter designations used routinelyin the art.

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are generally in the“L” isomeric form. Residues in the isomeric form can be substituted forany L-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxyl terminus of a polypeptide. In keeping withstandard polypeptide nomenclature described in J. Biol. Chem.,243:3557-59 (1968) and adopted at 37 C.F.R. §§ 1.821-1.822,abbreviations for amino acid residues are shown in Table 1:

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylalanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glutamic Acid and/or Glutamine W Trp TryptophanR Arg Arginine D Asp Aspartic acid N Asn Asparagine B Asx Aspartic Acidand/or Asparagine C Cys Cysteine X Xaa Unknown or other

All sequences of amino acid residues represented herein by a formulahave a left to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. In addition, the phrase “amino acidresidue” is defined to include the amino acids listed in the Table ofCorrespondence (Table 1), modified, non-natural and unusual amino acids.Furthermore, a dash at the beginning or end of an amino acid residuesequence indicates a peptide bond to a further sequence of one or moreamino acid residues or to an amino-terminal group such as NH₂ or to acarboxyl-terminal group such as COOH.

In a peptide or protein, suitable conservative substitutions of aminoacids are known to those of skill in this art and generally can be madewithout altering a biological activity of a resulting molecule. Those ofskill in this art recognize that in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.,Molecular Biology of the Gene, 4th Edition, 1987. The Benjamin/CummingsPub. co., p. 224).

Such substitutions can be made in accordance with the exemplarysubstitutions set forth in Table 2 as follows:

TABLE 2 Original residue Conservative substitution Ala (A) Gly; Ser Arg(R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G)Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T)Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu

Other substitutions also are permissible and can be determinedempirically or in accord with other known conservative ornon-conservative substitutions.

As used herein, “naturally occurring amino acids” refer to the 20L-amino acids that occur in polypeptides.

As used herein, the term “non-natural amino acid” refers to an organiccompound that has a structure similar to a natural amino acid but hasbeen modified structurally to mimic the structure and reactivity of anatural amino acid. Non-naturally occurring amino acids thus include,for example, amino acids or analogs of amino acids other than the 20naturally occurring amino acids and include, but are not limited to, theD-isostereomers of amino acids. Exemplary non-natural amino acids areknown to those of skill in the art, and include, but are not limited to,2-Aminoadipic acid (Aad), 3-Aminoadipic acid (Baad),β-alanine/β-Amino-propionic acid (Bala), 2-Aminobutyric acid (Abu),4-Aminobutyric acid/piperidinic acid (4Abu), 6-Aminocaproic acid (Acp),2-Aminoheptanoic acid (Ahe), 2-Aminoisobutyric acid (Aib),3-Aminoisobutyric acid (Baib), 2-Aminopimelic acid (Apm),2,4-Diaminobutyric acid (Dbu), Desmosine (Des), 2,2′-Diaminopimelic acid(Dpm), 2,3-Diaminopropionic acid (Dpr), N-Ethylglycine (EtGly),N-Ethylasparagine (EtAsn), Hydroxylysine (Hyl), allo-Hydroxylysine(Ahyl), 3-Hydroxyproline (3Hyp), 4-Hydroxyproline (4Hyp), Isodesmosine(Ide), alto-Isoleucine (Aile), N-Methylglycine, sarcosine (MeGly),N-Methylisoleucine (MeIle), 6-N-Methyllysine (MeLys), N-Methylvaline(MeVal), Norvaline (Nva), Norleucine (Nle), and Ornithine (Orn).

As used herein, a “native polypeptide” or a “native nucleic acid”molecule is a polypeptide or nucleic acid molecule, respectively, thatcan be found in nature. A native polypeptide or nucleic acid moleculecan be the wild-type form of a polypeptide or nucleic acid molecule. Anative polypeptide or nucleic acid molecule can be the predominant formof the polypeptide, or any allelic or other natural variant thereof. Thevariant polypeptides and nucleic acid molecules provided herein can havemodifications compared to native polypeptides and nucleic acidmolecules.

As used herein, the wild-type form of a polypeptide or nucleic acidmolecule is a form encoded by a gene or by a coding sequence encoded bythe gene. Typically, a wild-type form of a gene, or molecule encodedthereby, does not contain mutations or other modifications that alterfunction or structure. The term wild-type also encompasses forms withallelic variation as occurs among and between species. As used herein, apredominant form of a polypeptide or nucleic acid molecule refers to aform of the molecule that is the major form produced from a gene. A“predominant form” varies from source to source. For example, differentcells or tissue types can produce different forms of polypeptides, forexample, by alternative splicing and/or by alternative proteinprocessing. In each cell or tissue type, a different polypeptide can bea “predominant form.”

As used herein, an “allelic variant” or “allelic variation” referencesany of two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wild type form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species typicallyhave at least or about 80%, 85%, 90%, 95% or greater amino acid identitywith a wild type and/or predominant form from the same species; thedegree of identity depends upon the gene and whether comparison isinterspecies or intraspecies. Generally, intraspecies allelic variantshave at least or about 80%, 85%, 90% or 95% identity or greater with awild type and/or predominant form, including 96%, 97%, 98%, 99% orgreater identity with a wild type and/or predominant form of apolypeptide. Reference to an allelic variant herein generally refers tovariations n proteins among members of the same species.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude substitutions, deletions and insertions of nucleotides. Anallele of a gene also can be a form of a gene containing a mutation.

As used herein, “species variants” refer to variants in polypeptidesamong different species, including different mammalian species, such asmouse and human, and species of microorganisms, such as viruses andbacteria.

As used herein, a polypeptide “domain” is a part of a polypeptide (asequence of three or more, generally 5, 10 or more amino acids) that isa structurally and/or functionally distinguishable or definable.Exemplary of a polypeptide domain is a part of the polypeptide that canform an independently folded structure within a polypeptide made up ofone or more structural motifs (e.g. combinations of alpha helices and/orbeta strands connected by loop regions) and/or that is recognized by aparticular functional activity, such as enzymatic activity, dimerizationor antigen-binding. A polypeptide can have one or more, typically morethan one, distinct domains. For example, the polypeptide can have one ormore structural domains and one or more functional domains. A singlepolypeptide domain can be distinguished based on structure and function.A domain can encompass a contiguous linear sequence of amino acids.Alternatively, a domain can encompass a plurality of non-contiguousamino acid portions, which are non-contiguous along the linear sequenceof amino acids of the polypeptide. Typically, a polypeptide contains aplurality of domains. For example, each heavy chain and each light chainof an antibody molecule contains a plurality of immunoglobulin (Ig)domains, each about 110 amino acids in length.

Those of skill in the art are familiar with polypeptide domains and canidentify them by virtue of structural and/or functional homology withother such domains. For exemplification herein, definitions areprovided, but it is understood that it is well within the skill in theart to recognize particular domains by name. If needed, appropriatesoftware can be employed to identify domains.

As used herein, a functional region of a polypeptide is a region of thepolypeptide that contains at least one functional domain (which impartsa particular function, such as an ability to interact with abiomolecule, for example, through antigen-binding, DNA binding, ligandbinding, or dimerization, or by enzymatic activity, for example, kinaseactivity or proteolytic activity); exemplary of functional regions ofpolypeptides are antibody domains, such as V_(H), V_(L), C_(H), C_(L),and portions thereof, such as CDRs, including CDR1, CDR2 and CDR3, orantigen-binding portions, such as antibody combining sites.

As used herein, a structural region of a polypeptide is a region of thepolypeptide that contains at least one structural domain.

As used herein, a “property” of a polypeptide, such as an antibody,refers to any property exhibited by a polypeptide, including, but notlimited to, binding specificity, structural configuration orconformation, protein stability, resistance to proteolysis,conformational stability, thermal tolerance, and tolerance to pHconditions. Changes in properties can alter an “activity” of thepolypeptide. For example, a change in the binding specificity of theantibody polypeptide can alter the ability to bind an antigen, and/orvarious binding activities, such as affinity or avidity, or in vivoactivities of the polypeptide.

As used herein, an “activity” or a “functional activity” of apolypeptide, such as an antibody, refers to any activity exhibited bythe polypeptide. Such activities can be empirically determined.Exemplary activities include, but are not limited to, ability tointeract with a biomolecule, for example, through antigen-binding, DNAbinding, ligand binding, or dimerization, enzymatic activity, forexample, kinase activity or proteolytic activity. For an antibody(including antibody fragments), activities include, but are not limitedto, the ability to specifically bind a particular antigen, affinity ofantigen-binding (e.g. high or low affinity), avidity of antigen-binding(e.g. high or low avidity), on-rate, off-rate, effector functions, suchas the ability to promote antigen neutralization or clearance, virusneutralization, and in vivo activities, such as the ability to preventinfection or invasion of a pathogen, or to promote clearance, or topenetrate a particular tissue or fluid or cell in the body. Activity canbe assessed in vitro or in vivo using recognized assays, such as ELISA,flow cytometry, surface plasmon resonance or equivalent assays tomeasure on- or off-rate, immunohistochemistry and immunofluorescencehistology and microscopy, cell-based assays, flow cytometry and bindingassays (e.g., panning assays). For example, for an antibody polypeptide,activities can be assessed by measuring binding affinities, avidities,and/or binding coefficients (e.g., for on-/off-rates), and otheractivities in vitro or by measuring various effects in vivo, such asimmune effects, e.g. antigen clearance, penetration or localization ofthe antibody into tissues, protection from disease, e.g. infection,serum or other fluid antibody titers, or other assays that are wellknown in the art. The results of such assays that indicate that apolypeptide exhibits an activity can be correlated to activity of thepolo peptide in vivo, in which in vivo activity can be referred to astherapeutic activity, or biological activity. Activity of a modifiedpolypeptide can be any level of percentage of activity of the unmodifiedpolypeptide, including but not limited to, 1% of the activity, 2%, 3%,4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more ofactivity compared to the unmodified polypeptide. Assays to determinefunctionality or activity of modified (e.g. variant) antibodies are wellknown in the art.

As used herein, “therapeutic activity” refers to the in vivo activity ofa therapeutic polypeptide. Generally, the therapeutic activity is theactivity that is used to treat a disease or condition. Therapeuticactivity of a modified polypeptide can be any level of percentage oftherapeutic activity of the unmodified polypeptide, including but notlimited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,200%, 300%, 400%, 500%, or more of therapeutic activity compared to theunmodified polypeptide.

As used herein, “exhibits at least one activity” or “retains at leastone activity” refers to the activity exhibited by a modifiedpolypeptide, such as a variant polypeptide produced according to theprovided methods, such as a modified, e.g. variant antibody or othertherapeutic polypeptide (e.g. a modified anti-RSV antibody orantigen-binding fragment thereof), compared to the target or unmodifiedpolypeptide, that does not contain the modification. A modified, orvariant, polypeptide that retains an activity of a target polypeptidecan exhibit improved activity or maintain the activity of the unmodifiedpolypeptide. In some instances, a modified, or variant, polypeptide canretain an activity that is increased compared to an target or unmodifiedpolypeptide. In some cases, a modified, or variant, polypeptide canretain an activity that is decreased compared to an unmodified or targetpolypeptide. Activity of a modified, or variant, polypeptide can be anylevel of percentage of activity of the unmodified or target polypeptide,including but not limited to, 1% of the activity, 2%, 3%, 4%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, or more activity comparedto the unmodified or target polypeptide. In other embodiments, thechange in activity is at least about 2 times, 3 times, 4 times, 5 times,6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times,900 times, 1000 times, or more times greater than unmodified or targetpolypeptide. Assays for retention of an activity depend on the activityto be retained. Such assays can be performed in vitro or in vivo.Activity can be measured, for example, using assays known in the art anddescribed in the Examples below for activities such as but not limitedto ELISA and panning assays. Activities of a modified, or variant,polypeptide compared to an unmodified or target polypeptide also can beassessed in terms of an in vivo therapeutic or biological activity orresult following administration of the polypeptide.

As used herein, the term “assessing” is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of a protease, or a domain thereof, present inthe sample, and also of obtaining an index, ratio, percentage, visual,or other value indicative of the level of the activity. Assessment canbe direct or indirect and the chemical species actually detected neednot of course be the proteolysis product itself but can for example be aderivative thereof or some further substance. For example, detection ofa cleavage product of a complement protein, such as by SDS-PAGE andprotein staining with Coomassie blue.

As used herein, “nucleic acid” refers to at least two linked nucleotidesor nucleotide derivatives, including a deoxyribonucleic acid (DNA) and aribonucleic acid (RNA), joined together, typically by phosphodiesterlinkages. Also included in the term “nucleic acid” are analogs ofnucleic acids such as peptide nucleic acid (PNA). phosphorothioate DNA,and other such analogs and derivatives or combinations thereof. Nucleicacids also include DNA and RNA derivatives containing, for example, anucleotide analog or a “backbone” bond other than a phosphodiester bond,for example, a phosphotriester bond, a phosphoramidate bond, aphosphorothioate bond, a thioester bond, or a peptide bond (peptidenucleic acid). The term also includes, as equivalents, derivatives,variants and analogs of either RNA or DNA made from nucleotide analogs,single (sense or antisense) and double-stranded nucleic acids.Deoxyribonucleotides include deoxyadenosine, deoxycytidine,deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.

Nucleic acids can contain nucleotide analogs, including, for example,mass modified nucleotides, which allow for mass differentiation ofnucleic acid molecules; nucleotides containing a detectable label suchas a fluorescent, radioactive, luminescent or chemiluminescent label,which allow for detection of a nucleic acid molecule; or nucleotidescontaining a reactive group such as biotin or a thiol group, whichfacilitates immobilization of a nucleic acid molecule to a solidsupport. A nucleic acid also can contain one or more backbone bonds thatare selectively cleavable, for example, chemically, enzymatically orphotolytically cleavable. For example, a nucleic acid can include one ormore deoxyribonucleotides, followed by one or more ribonucleotides,which can be followed by one or more deoxyribonucleotides, such asequence being cleavable at the ribonucleotide sequence by basehydrolysis. A nucleic acid also can contain one or more bonds that arerelatively resistant to cleavage, for example, a chimericoligonucleotide primer, which can include nucleotides linked by peptidenucleic acid bonds and at least one nucleotide at the 3′ end, which islinked by a phosphodiester bond or other suitable bond, and is capableof being extended by a polymerase. Peptide nucleic acid sequences can beprepared using well-known methods (see, for example, Weiler et al.(1997) Nucleic Acids Res. 25:2792-2799).

As used herein, the terms “polynucleotide” and “nucleic acid molecule”refer to an oligomer or polymer containing at least two linkednucleotides or nucleotide derivatives, including a deoxyribonucleic acid(DNA) and a ribonucleic acid (RNA), joined together, typically byphosphodiester linkages. Polynucleotides also include DNA and RNAderivatives containing, for example, a nucleotide analog or a “backbone”bond other than a phosphodiester bond, for example, a phosphotriesterbond, a phosphoramidate bond, a phosphorothioate bond, a thioester bond,or a peptide bond (peptide nucleic acid). Polynucleotides (nucleic acidmolecules), include single-stranded and/or double-strandedpolynucleotides, such as deoxyribonucleic acid (DNA), and ribonucleicacid (RNA) as well as analogs or derivatives of either RNA or DNA. Theterm also includes, as equivalents, derivatives, variants and analogs ofeither RNA or DNA made from nucleotide analogs, single (sense orantisense) and double-stranded polynucleotides. Deoxyribonucleotidesinclude deoxyadenosine, deoxycytidine, deoxyguanosine anddeoxythymidine. For RNA, the uracil base is uridine. Polynucleotides cancontain nucleotide analogs, including, for example, mass modifiednucleotides, which allow for mass differentiation of polynucleotides;nucleotides containing a detectable label such as a fluorescent,radioactive, luminescent or chemiluminescent label, which allow fordetection of a polynucleotide; or nucleotides containing a reactivegroup such as biotin or a thiol group, which facilitates immobilizationof a polynucleotide to a solid support. A polynucleotide also cancontain one or more backbone bonds that are selectively cleavable, forexample, chemically, enzymatically or photolytically cleavable. Forexample, a polynucleotide can include one or more deoxyribonucleotides,followed by one or more ribonucleotides, which can be followed by one ormore deoxyribonucleotides, such a sequence being cleavable at theribonucleotide sequence by base hydrolysis. A polynucleotide also cancontain one or more bonds that are relatively resistant to cleavage, forexample, a chimeric oligonucleotide primer, which can includenucleotides linked by peptide nucleic acid bonds and at least onenucleotide at the 3′ end, which is linked by a phosphodiester bond orother suitable bond, and is capable of being extended by a polymerase.Peptide nucleic acid sequences can be prepared using well-known methods(see, for example, Weiler et al. (1997) Nucleic Acids Res.25:2792-2799). Exemplary of the nucleic acid molecules (polynucleotides)provided herein are oligonucleotides, including syntheticoligonucleotides, oligonucleotide duplexes, primers, including fill-inprimers, and oligonucleotide duplex cassettes.

As used herein, a “DNA construct” is a single or double stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a “DNA segment” is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, a positive strand polynucleotide refers to the “sensestrand” or a polynucleotide duplex, which is complementary to thenegative strand or the “antisense” strand. In the case ofpolynucleotides which encode genes, the sense strand is the strand thatis identical to the mRNA strand that is translated into a polypeptide,while the antisense strand is complementary to that strand. Positive andnegative strands of a duplex are complementary to one another.

As used herein, a genetic element refers to a gene, or any regionthereof, that encodes a polypeptide or protein or region thereof. Insome examples, a genetic element encodes a fusion protein.

As used herein, regulatory region of a nucleic acid molecule means acis-acting nucleotide sequence that influences expression, positively ornegatively, of an operatively linked gene. Regulatory regions includesequences of nucleotides that confer inducible (i.e., require asubstance or stimulus for increased transcription) expression of a gene.When an inducer is present or at increased concentration, geneexpression can be increased. Regulatory regions also include sequencesthat confer repression of gene expression (i.e., a substance or stimulusdecreases transcription). When a repressor is present or at increasedconcentration gene expression can be decreased. Regulatory regions areknown to influence, modulate or control many in vivo biologicalactivities including cell proliferation, cell growth and death, celldifferentiation and immune modulation. Regulatory regions typically bindto one or more trans-acting proteins, which results in either increasedor decreased transcription of the gene.

Particular examples of gene regulatory regions are promoters andenhancers. Promoters are sequences located around the transcription ortranslation start site, typically positioned 5′ of the translation startsite. Promoters usually are located within 1 Kb of the translation startsite, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5Kb or more, up to and including 10 Kb. Enhancers are known to influencegene expression when positioned 5′ or 3′ of the gene, or when positionedin or a part of an exon or an intron. Enhancers also can function at asignificant distance from the gene, for example, at a distance fromabout 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

Regulatory regions also include, but are not limited to, in addition topromoter regions, sequences that facilitate translation, splicingsignals for introns, maintenance of the correct reading frame of thegene to permit in-frame translation of mRNA and, stop codons, leadersequences and fusion partner sequences, internal ribosome binding site(IRES) elements for the creation of multigene, or polycistronic,messages, polyadenylation signals to provide proper polyadenylation ofthe transcript of a gene of interest and stop codons, and can beoptionally included in an expression vector.

As used herein, “operably linked” with reference to nucleic acidsequences, regions, elements or domains means that the nucleic acidregions are functionally related to each other. For example, nucleicacid encoding a leader peptide can be operably linked to nucleic acidencoding a polypeptide, whereby the nucleic acids can be transcribed andtranslated to express a functional fusion protein, wherein the leaderpeptide effects secretion of the fusion polypeptide. In some instances,the nucleic acid encoding a first polypeptide (e.g., a leader peptide)is operably linked to nucleic acid encoding a second polypeptide and thenucleic acids are transcribed as a single mRNA transcript, buttranslation of the mRNA transcript can result in one of two polypeptidesbeing expressed. For example, an amber stop codon can be located betweenthe nucleic acid encoding the first polypeptide and the nucleic acidencoding the second polypeptide, such that, when introduced into apartial amber suppressor cell, the resulting single mRNA transcript canbe translated to produce either a fusion protein containing the firstand second polypeptides, or can be translated to produce only the firstpolypeptide. In another example, a promoter can be operably linked tonucleic acid encoding a polypeptide, whereby the promoter regulates ormediates the transcription of the nucleic acid.

As used herein, “synthetic,” with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, “expression” refers to the process by which polypeptidesare produced by transcription and translation of polynucleotides. Thelevel of expression of a polypeptide can be assessed using any methodknown in art, including, for example, methods of determining the amountof the polypeptide produced from the host cell. Such methods caninclude, but are not limited to, quantitation of the polypeptide in thecell lysate by ELISA, Coomassie blue staining following gelelectrophoresis, Lowry protein assay and Bradford protein assay.

As used herein, a “host cell” is a cell that is used in to receive,maintain, reproduce and amplify a vector. A host cell also can be usedto express the polypeptide encoded by the vector. The nucleic acidcontained in the vector is replicated when the host cell divides,thereby amplifying the nucleic acids. In one example, the host cell is agenetic package, which can be induced to express the variant polypeptideon its surface. In another example, the host cell is infected with thegenetic package. For example, the host cells can be phage-displaycompatible host cells, which can be transformed with phage or phagemidvectors and accommodate the packaging of phage expressing fusionproteins containing the variant polypeptides.

As used herein, a “vector” is a replicable nucleic acid from which oneor more heterologous proteins can be expressed when the vector istransformed into an appropriate host cell. Reference to a vectorincludes those vectors into which a nucleic acid encoding a polypeptideor fragment thereof can be introduced, typically by restriction digestand ligation. Reference to a vector also includes those vectors thatcontain nucleic acid encoding a polypeptide. The vector is used tointroduce the nucleic acid encoding the polypeptide into the host cellfor amplification of the nucleic acid or for expression/display of thepolypeptide encoded by the nucleic acid. The vectors typically remainepisomal, but can be designed to effect integration of a gene or portionthereof into a chromosome of the genome. Also contemplated are vectorsthat are artificial chromosomes, such as yeast artificial chromosomesand mammalian artificial chromosomes. Selection and use of such vehiclesare well known to those of skill in the art.

As used herein, a vector also includes “virus vectors” or “viralvectors.” Viral vectors are engineered viruses that are operativelylinked to exogenous genes to transfer (as vehicles or shuttles) theexogenous genes into cells.

As used herein, an “expression vector” includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal, and the like. Expression vectors are generallyderived from plasmid or viral DNA, or can contain elements of both.Thus, an expression vector refers to a recombinant DNA or RNA construct,such as a plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein, the terms “oligonucleotide” and “oligo” are usedsynonymously. Oligonucleotides are polynucleotides that contain alimited number of nucleotides in length. Those in the art recognize thatoligonucleotides generally are less than at or about two hundred fifty,typically less than at or about two hundred, typically less than at orabout one hundred, nucleotides in length. Typically, theoligonucleotides provided herein are synthetic oligonucleotides. Thesynthetic oligonucleotides contain fewer than at or about 250 or 200nucleotides in length, for example, fewer than about 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200nucleotides in length. Typically, the oligonucleotides aresingle-stranded oligonucleotides. The ending “mer” can be used to denotethe length of an oligonucleotide. For example, “100-mer” can be used torefer to an oligonucleotide containing 100 nucleotides in length.Exemplary of the synthetic oligonucleotides provided herein are positiveand negative strand oligonucleotides, randomized oligonucleotides,reference sequence oligonucleotides, template oligonucleotides andfill-in primers are.

As used herein, synthetic oligonucleotides are oligonucleotides producedby chemical synthesis. Chemical oligonucleotide synthesis methods arewell known. Any of the known synthesis methods can be used to producethe oligonucleotides designed and used in the provided methods. Forexample, synthetic oligonucleotides typically are made by chemicallyjoining single nucleotide monomers or nucleotide trimers containingprotective groups. Typically, phosphoramidites, single nucleotidescontaining protective groups are added one at a time. Synthesistypically begins with the 3′ end of the oligonucleotide. The 3′ mostphosphoramidite is attached to a solid support and synthesis proceeds byadding each phosphoramidite to the 5′ end of the last. After eachaddition, the protective group is removed from the 5′ phosphate group onthe most recently added base, allowing addition of anotherphosphoramidite. Automated synthesizers generally can synthesizeoligonucleotides up to about 150 to about 200 nucleotides in length.Typically, the oligonucleotides designed and used in the providedmethods are synthesized using standard cyanoethyl chemistry fromphosphoramidite monomers. Synthetic oligonucleotides produced by thisstandard method can be purchased from Integrated DNA Technologies (IDT)(Coralville, Iowa) or TriLink Biotechnologies (San Diego, Calif.).

As used herein, “primer” refers to a nucleic acid molecule (moretypically, to a pool of such molecules sharing sequence identity) thatcan act as a point of initiation of template-directed nucleic acidsynthesis under appropriate conditions (for example, in the presence offour different nucleoside triphosphates and a polymerization agent, suchas DNA polymerase. RNA polymerase or reverse transcriptase) in anappropriate buffer and at a suitable temperature. It will be appreciatedthat certain nucleic acid molecules can serve as a “probe” and as a“primer.” A primer, however, has a 3′ hydroxyl group for extension. Aprimer can be used in a variety of methods, including, for example,polymerase chain reaction (PCR), reverse-transcriptase (RT)-PCR, RNAPCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′and 5′ RACE, in situ PCR, ligation-mediated PCR and other amplificationprotocols.

As used herein, “primer pair” refers to a set of primers (e.g. two poolsof primers) that includes a 5′ (upstream) primer that specificallyhybridizes with the 5′ end of a sequence to be amplified (e.g. by PCR)and a 3′ (downstream) primer that specifically hybridizes with thecomplement of the 3′ end of the sequence to be amplified. Because“primer” can refer to a pool of identical nucleic acid molecules, aprimer pair typically is a pair of two pools of primers.

As used herein, “single primer” and “single primer pool” refersynonymously to a pool of primers, where each primer in the poolcontains sequence identity with the other primer members, for example, apool of primers where the members share at least at or about 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity. The primersin the single primer pool (all sharing sequence identity) act as 5′(upstream) primers (that specifically hybridize with the 5′ end of asequence to be amplified (e.g. by PCR)) and as 3′ (downstream) primers(that specifically hybridize with the complement of the 3′ end of thesequence to be amplified). Thus, the single primer can be used, withoutother primers, to prime synthesis of complementary strands and amplify anucleic acid in a polymerase amplification reaction.

As used herein, complementarity, with respect to two nucleotides, refersto the ability of the two nucleotides to base pair with one another uponhybridization of two nucleic acid molecules. Two nucleic acid moleculessharing complementarity are referred to as complementary nucleic acidmolecules; exemplary of complementary nucleic acid molecules are thepositive and negative strands in a polynucleotide duplex. As usedherein, when a nucleic acid molecule or region thereof is complementaryto another nucleic acid molecule or region thereof, the two molecules orregions specifically hybridize to each other. Two complementary nucleicacid molecules can be described in terms of percent complementarity. Forexample, two nucleic acid molecules, each 100 nucleotides in length,that specifically hybridize with one another but contain 5 mismatcheswith respect to one another, are said to be 95% complementary. For twonucleic acid molecules to hybridize with 100% complementarity, it is notnecessary that complementarity exist along the entire length of both ofthe molecules. For example, a nucleic acid molecule containing 20contiguous nucleotides in length can specifically hybridize to acontiguous 20 nucleotide portion of a nucleic acid molecule containing500 contiguous nucleotide in length. If no mismatches occur along this20 nucleotide portion, the 20 nucleotide molecule hybridizes with 100%complementarity. Typically, complementary nucleic acid molecules alignwith less than 25%, 20%, 15%, 10%, 5% 4%, 3%, 2% or 1% mismatchesbetween the complementary nucleotides (in other words, at least at orabout 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% complementarity). Inanother example, the complementary nucleic acid molecules contain at orabout or at least at or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% complementarity. In one example,complementary nucleic acid molecules contain fewer than 5, 4, 3, 2 or 1mismatched nucleotides. In one example, the complementary nucleotidesare 100% complementary. If necessary, the percentage of complementaritywill be specified. Typically the two molecules are selected such thatthey will specifically hybridize under conditions of high stringency.

As used herein, a complementary strand of a nucleic acid molecule refersto a sequence of nucleotides, e.g., a nucleic acid molecule, thatspecifically hybridizes to the molecule, such as the opposite strand tothe nucleic acid molecule in a polynucleotide duplex. For example, in apolynucleotide duplex, the complementary strand of a positive strandoligonucleotide is a negative strand oligonucleotide that specificallyhybridizes to the positive strand oligonucleotide in a duplex. In oneexample of the provided methods, polymerase reactions are used tosynthesize complementary strands of polynucleotides to form duplexes,typically beginning by hybridizing an oligonucleotide primer to thepolynucleotide.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide or polynucleotide) to another nucleic acid molecule.Those of skill in the art are familiar with in vitro and in vivoparameters that affect specific hybridization, such as length andcomposition of the particular molecule. Parameters particularly relevantto in vitro hybridization further include annealing and washingtemperature, buffer composition and salt concentration. It is notnecessary that two nucleic acid molecules exhibit 100% complementarityin order to specifically hybridize to one another. For example, twocomplementary nucleic acid molecules sharing sequence complementarity,such as at or about or at least or about 99%, 98%, 97%, 96%, 95%, 90%,85%, 80%, 75%, 70%, 65%, 60%, 55% or 50% complementarity, canspecifically hybridize to one another. Parameters, for example, buffercomponents, time and temperature, used in in vitro hybridization methodsprovided herein, can be adjusted in stringency to vary the percentcomplementarity required for specific hybridization of two nucleic acidmolecules. The skilled person can readily adjust these parameters toachieve specific hybridization of a nucleic acid molecule to a targetnucleic acid molecule appropriate for a particular application.

As used herein, “primary sequence” refers to the sequence of amino acidresidues in a polypeptide or the sequence of nucleotides in a nucleicacid molecule.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the sequence of amino acids of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity of sequences of residues and theresidues contained therein. Methods for assessing the degree ofsimilarity between proteins or nucleic acids are known to those of skillin the art. For example, in one method of assessing sequence similarity,two amino acid or nucleotide sequences are aligned in a manner thatyields a maximal level of identity between the sequences. “Identity”refers to the extent to which the amino acid or nucleotide sequences areinvariant. Alignment of amino acid sequences, and to some extentnucleotide sequences, also can take into account conservativedifferences and/or frequent substitutions in amino acids (ornucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

As used herein, when a polypeptide or nucleic acid molecule or regionthereof contains or has “identity” or “homology” to another polypeptideor nucleic acid molecule or region, the two molecules and/or regionsshare greater than or equal to at or about 40% sequence identity, andtypically greater than or equal to at or about 50% sequence identity,such as at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% sequence identity; the precise percentage ofidentity can be specified if necessary. A nucleic acid molecule, orregion thereof that is identical or homologous to a second nucleic acidmolecule or region can specifically hybridize to a nucleic acid moleculeor region that is 100% complementary to the second nucleic acid moleculeor region. Identity alternatively can be compared between twotheoretical nucleotide or amino acid sequences or between a nucleic acidor polypeptide molecule and a theoretical sequence.

Sequence “identity,” per se, has an art-recognized meaning and thepercentage of sequence identity between two nucleic acid or polypeptidemolecules or regions can be calculated using published techniques.Sequence identity can be measured along the full length of apolynucleotide or polypeptide or along a region of the molecule. (See,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M, and Devereux, J., eds., M Stockton Press, New York,1991). While there exist a number of methods to measure identity betweentwo polynucleotide or polypeptides, the term “identity” is well known toskilled artisans (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073(1988)).

Sequence identity compared along the full length of two polynucleotidesor polypeptides refers to the percentage of identical nucleotide oramino acid residues along the full-length of the molecule. For example,if a polypeptide A has 100 amino acids and polypeptide B has 95 aminoacids, which are identical to amino acids 1-95 of polypeptide A, thenpolypeptide B has 95% identity when sequence identity is compared alongthe full length of a polypeptide A compared to full length ofpolypeptide B. Alternatively, sequence identity between polypeptide Aand polypeptide B can be compared along a region, such as a 20 aminoacid analogous region, of each polypeptide. In this case, if polypeptideA and B have 20 identical amino acids along that region, the sequenceidentity for the regions is 100%. Alternatively, sequence identity canbe compared along the length of a molecule, compared to a region ofanother molecule. Alternatively, sequence identity between polypeptide Aand polypeptide B can be compared along the same length polypeptide butwith amino acid replacements, such as conservative amino acidreplacements or non-conservative amino acid replacements. As discussedbelow, and known to those of skill in the art, various programs andmethods for assessing identity are known to those of skill in the art.High levels of identity, such as 90% or 95% identity, readily can bedetermined without software.

Whether any two nucleic acid molecules have nucleotide sequences thatare at least or about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%“identical” can be determined using known computer algorithms such asthe “FASTA” program, using for example, the default parameters as inPearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programsinclude the GCG program package (Devereux, J. et al. (1984) NucleicAcids Research 12(1):387), BLASTP, BLASTN, FASTA (Altschul, S. F. et al.(1990) J. Molec. Biol. 215:403; Guide to Huge Computers, Martin J.Bishop, ed., Academic Press, San Diego, 1994, and Carrillo et al. (1988)SIAM J Applied Math 48:1073). For example, the BLAST function of theNational Center for Biotechnology Information database can be used todetermine identity. Other commercially or publicly available programsinclude. DNAStar “MegAlign” program (Madison, Wis.) and the Universityof Wisconsin Genetics Computer Group (UWG) “Gap” program (MadisonWis.)). Percent homology or identity of proteins and/or nucleic acidmolecules can be determined, for example, by comparing sequenceinformation using a GAP computer program (e.g., Needleman et al. (1970)J. Mol. Biol. 48:443, as revised by Smith and Waterman ((1981) Adv.Appl. Math. 2:482). Briefly, the GAP program defines similarity as thenumber of aligned symbols (i.e., nucleotides or amino acids), which aresimilar, divided by the total number of symbols in the shorter of thetwo sequences. Default parameters for the GAP program can include: (1) aunary comparison matrix (containing a value of 1 for identities and 0for non-identities) and the weighted comparison matrix of Gribskov etal. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz andDayhoff, eds., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, NationalBiomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps.

In general, for determination of the percentage sequence identity,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M, and Devereux, J., eds., M Stockton Press, New York,1991; Carrillo et al. (1988) SIAM J Applied Math 48:1073). For sequenceidentity, the number of conserved amino acids is determined by standardalignment algorithms programs, and can be used with default gappenalties established by each supplier. Substantially homologous nucleicacid molecules specifically hybridize typically at moderate stringencyor at high stringency all along the length of the nucleic acid ofinterest. Also contemplated are nucleic acid molecules that containdegenerate codons in place of codons in the hybridizing nucleic acidmolecule.

Therefore, the term “identity,” when associated with a particularnumber, represents a comparison between the sequences of a first and asecond polypeptide or polynucleotide or regions thereof and/or betweentheoretical nucleotide or amino acid sequences. As used herein, the termat least “90% identical to” refers to percent identities from 90 to99.99 relative to the first nucleic acid or amino acid sequence of thepolypeptide. Identity at a level of 90% or more is indicative of thefact that, assuming for exemplification purposes, a first and secondpolypeptide length of 100 amino acids are compared, no more than 10%(i.e., 10 out of 100) of the amino acids in the first polypeptidediffers from that of the second polypeptide. Similar comparisons can bemade between first and second polynucleotides. Such differences amongthe first and second sequences can be represented as point mutationsrandomly distributed over the entire length of a polypeptide or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g. 10/100 amino acid difference (approximately 90%identity). Differences are defined as nucleotide or amino acid residuesubstitutions, insertions, additions or deletions. At the level ofhomologies or identities above about 85-90%, the result is independentof the program and gap parameters set; such high levels of identity canbe assessed readily, often by manual alignment without relying onsoftware.

As used herein, alignment of a sequence refers to the use of homology toalign two or more sequences of nucleotides or amino acids. Typically,two or more sequences that are related by 50% or more identity arealigned. An aligned set of sequences refers to 2 or more sequences thatare aligned at corresponding positions and can include aligningsequences derived from RNAs, such as ESTs and other cDNAs, aligned withgenomic DNA sequence.

Related or variant polypeptides or nucleic acid molecules can be alignedby any method known to those of skill in the art. Such methods typicallymaximize matches, and include methods, such as using manual alignmentsand by using the numerous alignment programs available (e.g., BLASTP)and others known to those of skill in the art. By aligning the sequencesof polypeptides or nucleic acids, one skilled in the art can identifyanalogous portions or positions, using conserved and identical aminoacid residues as guides. Further, one skilled in the art also can employconserved amino acid or nucleotide residues as guides to findcorresponding amino acid or nucleotide residues between and among humanand non-human sequences. Corresponding positions also can be based onstructural alignments, for example by using computer simulatedalignments of protein structure. In other instances, correspondingregions can be identified. One skilled in the art also can employconserved amino acid residues as guides to find corresponding amino acidresidues between and among human and non-human sequences.

As used herein, “analogous” and “corresponding” portions, positions orregions are portions, positions or regions that are aligned with oneanother upon aligning two or more related polypeptide or nucleic acidsequences (including sequences of molecules, regions of molecules and/ortheoretical sequences) so that the highest order match is obtained,using an alignment method known to those of skill in the art to maximizematches. In other words, two analogous positions (or portions orregions) align upon best-fit alignment of two or more polypeptide ornucleic acid sequences. The analogous portions/positions/regions areidentified based on position along the linear nucleic acid or amino acidsequence when the two or more sequences are aligned. The analogousportions need not share any sequence similarity with one another. Forexample, alignment (such that maximizing matches) of the sequences oftwo homologous nucleic acid molecules, each 100 nucleotides in length,can reveal that 70 of the 100 nucleotides are identical. Portions ofthese nucleic acid molecules containing some or all of the othernon-identical 30 amino acids are analogous portions that do not sharesequence identity. Alternatively, the analogous portions can containsome percentage of sequence identity to one another, such as at or about50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, orfractions thereof. In one example, the analogous portions are 100%identical.

As used herein, a “modification” is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements of amino acids and nucleotides, respectively. Methods ofmodifying a polypeptide are routine to those of skill in the art, suchas by using recombinant DNA methodologies.

As used herein, “deletion,” when referring to a nucleic acid orpolypeptide sequence, refers to the deletion of one or more nucleotidesor amino acids compared to a sequence, such as a target polynucleotideor polypeptide or a native or wild-type sequence.

As used herein, “insertion” when referring to a nucleic acid or aminoacid sequence, describes the inclusion of one or more additionalnucleotides or amino acids, within a target, native, wild-type or otherrelated sequence. Thus, a nucleic acid molecule that contains one ormore insertions compared to a wild-type sequence, contains one or moreadditional nucleotides within the linear length of the sequence. As usedherein, “additions,” to nucleic acid and amino acid sequences describeaddition of nucleotides or amino acids onto either termini compared toanother sequence.

As used herein, “substitution” refers to the replacing of one or morenucleotides or amino acids in a native, target, wild-type or othernucleic acid or polypeptide sequence with an alternative nucleotide oramino acid, without changing the length (as described in numbers ofresidues) of the molecule. Thus, one or more substitutions in a moleculedoes not change the number of amino acid residues or nucleotides of themolecule. Substitution mutations compared to a particular polypeptidecan be expressed in terms of the number of the amino acid residue alongthe length of the polypeptide sequence. For example, a modifiedpolypeptide having a modification in the amino acid at the 19^(th)position of the amino acid sequence that is a substitution of Isoleucine(Ile; I) for cysteine (Cys: C) can be expressed as 119C, Ile19C, orsimply C19, to indicate that the amino acid at the modified 19^(1h)position is a cysteine. In this example, the molecule having thesubstitution has a modification at Ile 19 of the unmodified polypeptide.

As used herein, a binding property is a characteristic of a molecule,e.g. a polypeptide, relating to whether or not, and how, it binds one ormore binding partners. Binding properties include ability to bind thebinding partner(s), the affinity with which it binds to the bindingpartner (e.g. high affinity), the avidity with which it binds to thebinding partner, the strength of the bond with the binding partner andspecificity for binding with the binding partner.

As used herein, affinity describes the strength of the interactionbetween two or more molecules, such as binding partners, typically thestrength of the noncovalent interactions between two binding partners.The affinity of an antibody or antigen-binding fragment thereof for anantigen epitope is the measure of the strength of the total noncovalentinteractions between a single antibody combining site and the epitope.Low-affinity antibody-antigen interaction is weak, and the moleculestend to dissociate rapidly, while high affinity antibody-antigen-bindingis strong and the molecules remain bound for a longer amount of time.Methods for calculating affinity are well known, such as methods fordetermining association/dissociation constants. Affinity can beestimated empirically or affinities can be determined comparatively,e.g. by comparing the affinity of one antibody and another antibody fora particular antigen.

As used herein, antibody avidity refers to the strength of multipleinteractions between a multivalent antibody and its cognate antigen,such as with antibodies containing multiple binding sites associatedwith an antigen with repeating epitopes or an epitope array. A highavidity antibody has a higher strength of such interactions comparedwith a low avidity antibody.

As used herein, “bind” refers to the participation of a molecule in anyattractive interaction with another molecule, resulting in a stableassociation in which the two molecules are in close proximity to oneanother. Binding includes, but is not limited to, non-covalent bonds,covalent bonds (such as reversible and irreversible covalent bonds), andincludes interactions between molecules such as, but not limited to,proteins, nucleic acids, carbohydrates, lipids, and small molecules,such as chemical compounds including drugs. Exemplary of bonds areantibody-antigen interactions and receptor-ligand interactions. When anantibody “binds” a particular antigen, bind refers to the specificrecognition of the antigen by the antibody, through cognateantibody-antigen interaction, at antibody combining sites. Binding alsocan include association of multiple chains of a polypeptide, such asantibody chains which interact through disulfide bonds.

As used herein, “affinity constant” refers to an association constant(Ka) used to measure the affinity of an antibody for an antigen. Thehigher the affinity constant the greater the affinity of the antibodyfor the antigen. Affinity constants are expressed in units of reciprocalmolarity (i.e. M⁻¹) and can be calculated from the rate constant for theassociation-dissociation reaction as measured by standard kineticmethodology for antibody reactions (e.g., immunoassays, surface plasmonresonance, or other kinetic interaction assays known in the art).

As used herein, the term “the same,” when used in reference to antibodybinding affinity, means that the association constant (Ka) is withinabout 1 to 100 fold or 1 to 10 fold of the reference antibody (1-100fold greater affinity or 1-100 fold less affinity, or any numericalvalue or range or value within such ranges, than the referenceantibody).

As used herein, “substantially the same” when used in reference toassociation constant (Ka), means that the association constant is withinabout 5 to 5000 fold greater or less than the association constant, Ka,of the reference antibody (5-5000 fold greater or 5-5000 fold less thanthe reference antibody). The binding affinity of an antibody also can beexpressed as a dissociation constant, or Kd. The dissociation constantis the reciprocal of the association constant. Kd=1/Ka.

As used herein, the phase “having the same binding specificity” whenused to describe an antibody in reference to another antibody, meansthat the antibody specifically binds (immunospecifically binds orspecifically binds to the virus) to all or a part of the same antigenicepitope as the reference antibody. Thus, an anti-RSV antibody orantigen-binding fragment thereof having the same binding specificity asthe antibody denoted as 58c5 or as 30D8 specifically binds to all or apart of the same epitope as the anti-RSV antibody or antigen-bindingfragment thereof denoted as 58c5 or 30D8, respectively. The epitope canbe in the isolated protein, or in the protein in the virus. The abilityof two antibodies to bind to the same epitope can be determined by knownassays in the art such as, for example, surface plasmon resonance assaysand antibody competition assays. Typically, antibodies thatimmunospecifically bind to the same epitope can compete for binding tothe epitope, which can be measured, for example, by an in vitro bindingcompetition assay (e.g. competition ELISA), using techniques known theart. Typically, a first antibody that immunospecifically binds to thesame epitope as a second antibody can compete for binding to the epitopeby about or 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100%, where the percentage competition is measured ability ofthe second antibody to displace binding of the first antibody to theepitope. In exemplary competition assays, the antigen is incubated inthe presence a predetermined limiting dilution of a labeled antibody(e.g., 50-70% saturation concentration), and serial dilutions of anunlabeled competing antibody. Competition is determined by measuring thebinding of the labeled antibody to the antigen for any decreases inbinding in the presence of the competing antibody. Variations of suchassays, including various labeling techniques and detection methodsincluding, for example, radiometric, fluorescent, enzymatic andcolorimetric detection, are known in the art. The ability of a firstantibody to bind to the same epitope as a second antibody also can bedetermined, for example, by virus neutralization assays using MonoclonalAntibody-Resistant Mutants (MARMs). For example, where a first anti-RSVantibody neutralizes wild-type RSV but not a particular mutant RSV, asecond antibody that neutralizes the wild-type RSV but not theparticular mutant RSV generally binds the same epitope on RSV as thefirst antibody. Where a first anti-RSV antibody neutralizes wild-typeRSV but not a particular mutant RSV, a second antibody that neutralizesthe wild-type RSV and the particular mutant RSV generally does not bindthe same epitope on RSV as the first antibody.

As used herein, a “monoclonal antibody resistant mutant” (MARM) alsoreferred to as a “monoclonal antibody escape mutant” is a mutant RSVthat exhibits resistance to neutralization by a monoclonal antibody thatneutralizes the wildtype RSV virus. In practice, the concentration ofantibody needed to neutralize 50% of an RSV MARM must be at least aboutor at least 10-fold greater than that required to neutralize anequivalent amount of reference wild-type infectious virus particles forthe MARM to be considered to have escaped the antibody.

MARMs are generated by culturing wildtype RSV in the presence of suchmonoclonal antibody over successive rounds of viral replication in thepresence of the antibody such that after each successive round of virusreplication, increasing concentrations of antibody are required toproduce virus neutralization effects. Cytopathic effects (CPE) only areobserved in the presence of increasing concentrations of antibodiesuntil a mutant virus results that is no longer efficiently neutralizedby the antibody. If more rounds of replication are required for theemergence of a MARM in the presence of a first antibody compared to asecond antibody, one can conclude the first antibody binds to an epitopethat is different from the epitope to which the second antibody binds.If a first antibody can neutralize a MARM generated against a secondantibody, one can conclude that the antibodies specifically bind to orinteract with different epitopes. MARMs can more finely map the antigenbinding epitope of an antibody compared to a competition binding assay,such that one antibody can compete against another for binding to anantigen, but can s neutralize the MARM of its competitor. For someantibodies, it only requires a few rounds of selection to generate aMARM; for other antibodies more rounds are required. For certainantibodies provided herein, no MARMS are generated after 10, 11, 12, 13,14, 15, 20, 25, 30 or more rounds. In some instances, it is not possibleto generate a MARM.

As used herein, EC₅₀ refers to the effective concentration at which anantibody can inhibit virus infection 50% in an in vitro neutralizationassay, such as, for example, a virus plaque reduction assay as describedherein (e.g., a plaque reduction assay using Vero host cells or otherhost cell for infection) or other virus neutralization assays known inthe art. Typically, a neutralizing virus is one that has an EC₅₀ of 2 nMor less for inhibition of the virus in an in vitro neutralization assay,such as a virus plaque reduction assay.

As used herein, “binding partner” refers to a molecule (such as apolypeptide, lipid, glycolipid, nucleic acid molecule, carbohydrate orother molecule), with which another molecule specifically interacts, forexample, through covalent or noncovalent interactions, such as theinteraction of an antibody with cognate antigen. The binding partner canbe naturally or synthetically produced. In one example, desired variantpolypeptides are selected using one or more binding partners, forexample, using in vitro or in vivo methods. Exemplary of the in vitromethods include selection using a binding partner coupled to a solidsupport, such as a bead, plate, column, matrix or other solid support;or a binding partner coupled to another selectable molecule, such as abiotin molecule, followed by subsequent selection by coupling the otherselectable molecule to a solid support. Typically, the in vitro methodsinclude wash steps to remove unbound polypeptides, followed by elutionof the selected variant polypeptide(s). The process can be repeated oneor more times in an iterative process to select variant polypeptidesfrom among the selected polypeptides.

As used herein, a disulfide bond (also called an S—S bond or a disulfidebridge) is a single covalent bond derived from the coupling of thiolgroups. Disulfide bonds in proteins are formed between the thiol groupsof cysteine residues, and stabilize interactions between polypeptidedomains, such as antibody domains.

As used herein, “coupled” or “conjugated” means attached via a covalentor noncovalent interaction.

As used herein, the phrase “conjugated to an antibody” or “linked to anantibody” or grammatical variations thereof, when referring to theattachment of a moiety to an antibody or antigen-binding fragmentthereof, such as a diagnostic or therapeutic moiety, means that themoiety is attached to the antibody or antigen-binding fragment thereofby any known means for linking peptides, such as, for example, byproduction of fusion protein by recombinant means orpost-translationally by chemical means. Conjugation can employ any of avariety of linking agents to effect conjugation, including, but notlimited to, peptide or compound linkers or chemical cross-linkingagents.

As used herein, “phage display” refers to the expression of polypeptideson the surface of filamentous bacteriophage.

As used herein, a “phage-display compatible cell” or “phage-displaycompatible host cell” is a host cell, typically a bacterial host cell,that can be infected by phage and thus can support the production ofphage displaying fusion proteins containing polypeptides, e.g., variantpolypeptides and can thus be used for phage display. Exemplary of phagedisplay compatible cells include, but are not limited to, XL1-bluecells.

As used herein, “panning” refers to an affinity-based selectionprocedure for the isolation of phage displaying a molecule with aspecificity for a binding partner, for example, a capture molecule(e.g., an antigen) or sequence of amino acids or nucleotides or epitope,region, portion or locus therein.

As used herein, “display protein” or “genetic package display protein”means any genetic package polypeptide for display of a polypeptide onthe genetic package, such that when the display protein is fused to(e.g., included as part of a fusion protein with) a polypeptide ofinterest (e.g., a polypeptide for which reduced expression is desired),the polypeptide is displayed on the outer surface of the geneticpackage. The display protein typically is present on or within the outersurface or outer compartment of a genetic package (e.g., membrane, cellwall, coat or other outer surface or compartment) of a genetic package,e.g., a viral genetic package, such as a phage, such that upon fusion toa polypeptide of interest, the polypeptide is displayed on the geneticpackage.

As used herein, a coat protein is a display protein, at least a portionof which is present on the outer surface of the genetic package, suchthat when it is fused to the polypeptide of interest, the polypeptide isdisplayed on the outer surface of the genetic package. Typically, thecoat proteins are viral coat proteins, such as phage coat proteins. Aviral coat protein, such as a phage coat protein associates with thevirus particle during assembly in a host cell. In one example, coatproteins are used herein for display of polypeptides on geneticpackages; the coat proteins are expressed as portions of fusionproteins, which contain the coat protein sequence of amino acids and asequence of amino acids of the displayed polypeptide. The coat proteincan be a full-length coat protein or any portion thereof capable ofeffecting display of the polypeptide on the surface of the geneticpackage.

Exemplary of coat proteins are phage coat proteins, such as, but notlimited to, (i) minor coat proteins of filamentous phage, such as geneIII protein (gIIIp, cp3), and (ii) major coat proteins (which arepresent in the viral coat at 10 copies or more, for example, tens,hundreds or thousands of copies) of filamentous phage such as gene VIIIprotein (gVIIIp, cp8); fusions to other phage coat proteins such as geneVI protein, gene VII protein, or gene IX protein (see, e.g., WO00/71694); and portions (e.g., domains or fragments) of these proteins,such as, but not limited to domains that are stably incorporated intothe phage particle, e.g. such as the anchor domain of gIIIp, or gVIIIp.Additionally, mutants of gVIIIp can be used which are optimized forexpression of larger peptides, such as mutants having improved surfacedisplay properties, such as mutant gVIIp (see, for example, Sidhu et al.(2000) J. Mol. Biol. 296:487-495).

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms. Diseases and disorders ofinterest herein are those involving RSV infection or those that increasethe risk of a RSV infection.

As used herein, “infection” and “RSV infection” refer to all stages of aRSV life cycle in a host (including, but not limited to the invasion byand replication of RSV in a cell or body tissue), as well as thepathological state resulting from the invasion by and replication of aRSV. The invasion by and multiplication of a RSV includes, but is notlimited to, the following steps: the docking of the RSV particle to acell, fusion of a virus with a cell membrane, the introduction of viralgenetic information into a cell, the expression of RSV proteins, theproduction of new RSV particles and the release of RSV particles from acell. A RSV infection can be an upper respiratory tract RSV infection(URI), a lower respiratory tract RSV infection (LRI), or a combinationthereof. In some examples, the pathological state resulting from theinvasion by and replication of a RSV is an acute RSV disease.

As used herein, “acute RSV disease” refers to clinically significantdisease in the lungs or lower respiratory tract as a result of a RSVinfection, which can manifest as pneumonia and/or bronchiolitis, wheresuch symptoms can include, for example, hypoxia, apnea, respiratorydistress, rapid breathing, wheezing, and cyanosis. Acute RSV diseaserequires an affected individual to obtain medical intervention, such ashospitalization, administration of oxygen, intubation and/orventilation.

As used herein, “treating” a subject with a disease or condition meansthat the subject's symptoms are partially or totally alleviated, orremain static following treatment. Hence treatment encompassesprophylaxis, therapy and/or cure. Prophylaxis refers to prevention of apotential disease and/or a prevention of worsening of symptoms orprogression of a disease. Treatment also encompasses any pharmaceuticaluse of any antibody or antigen-binding fragment thereof provided orcompositions provided herein.

As used herein, “prevention” or prophylaxis, and grammaticallyequivalent forms thereof, refers to methods in which the risk ofdeveloping disease or condition is reduced.

As used herein, a “pharmaceutically effective agent” includes anytherapeutic agent or bioactive agents, including, but not limited to,for example, anesthetics, vasoconstrictors, dispersing agents,conventional therapeutic drugs, including small molecule drugs andtherapeutic proteins.

As used herein, a “therapeutic effect” means an effect resulting fromtreatment of a subject that alters, typically improves or amelioratesthe symptoms of a disease or condition or that cures a disease orcondition.

As used herein, a “therapeutically effective amount” or a“therapeutically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that is atleast sufficient to produce a therapeutic effect followingadministration to a subject. Hence, it is the quantity necessary forpreventing, curing, ameliorating, arresting or partially arresting asymptom of a disease or disorder.

As used herein, “therapeutic efficacy” refers to the ability of anagent, compound, material, or composition containing a compound toproduce a therapeutic effect in a subject to whom the an agent,compound, material, or composition containing a compound has beenadministered.

As used herein, a “prophylactically effective amount” or a“prophylactically effective dose” refers to the quantity of an agent,compound, material, or composition containing a compound that whenadministered to a subject, will have the intended prophylactic effect,e.g., preventing or delaying the onset, or reoccurrence, of disease orsymptoms, reducing the likelihood of the onset, or reoccurrence, ofdisease or symptoms, or reducing the incidence of viral infection. Thefull prophylactic effect does not necessarily occur by administration ofone dose, and can occur only after administration of a series of doses.Thus, a prophylactically effective amount can be administered in one ormore administrations.

As used herein, the terms “immunotherapeutically” or “immunotherapy” inconjunction with antibodies provided denotes prophylactic as well astherapeutic administration. Thus, the therapeutic antibodies providedcan be administered to a subject at risk of contracting a virusinfection (e.g. a RSV infection) in order to lessen the likelihoodand/or severity of the disease, or administered to subjects alreadyevidencing active virus infection (e.g. a RSV infection).

As used herein, amelioration of the symptoms of a particular disease ordisorder by a treatment, such as by administration of a pharmaceuticalcomposition or other therapeutic, refers to any lessening, whetherpermanent or temporary, lasting or transient, of the symptoms that canbe attributed to or associated with administration of the composition ortherapeutic.

As used herein, the term “diagnostically effective” amount refers to thequantity of an agent, compound, material, or composition containing adetectable compound that is at least sufficient for detection of thecompound following administration to a subject. Generally, adiagnostically effective amount of an anti-RSV antibody orantigen-binding fragment thereof, such as a detectably-labeled antibodyor antigen-binding fragment thereof or an antibody or antigen-bindingfragment thereof that can be detected by a secondary agent, administeredto a subject for detection is quantity of the antibody orantigen-binding fragment thereof which is sufficient to enable detectionof the site having the RSV antigen for which the antibody orantigen-binding fragment thereof is specific. In using the antibodiesprovided herein for the in vivo detection of antigen, a detectablylabeled antibody or antigen-binding fragment thereof is given in a dosewhich is diagnostically effective.

As used herein, a label or detectable moiety is a detectable marker(e.g., a fluorescent molecule, chemiluminescent molecule, abioluminescent molecule, a contrast agent (e.g., a metal), aradionuclide, a chromophore, a detectable peptide, or an enzyme thatcatalyzes the formation of a detectable product) that can be attached orlinked directly or indirectly to a molecule (e.g., an anti-RSV antibodyor antigen-binding fragment thereof provided herein) or associatedtherewith and can be detected in vivo and/or in vitro. The detectionmethod can be any method known in the art, including known in vivoand/or in vitro methods of detection (e.g., imaging by visualinspection, magnetic resonance (MR) spectroscopy, ultrasound signal,X-ray, gamma ray spectroscopy (e.g., positron emission tomography (PET)scanning, single-photon emission computed tomography (SPECT)),fluorescence spectroscopy or absorption). Indirect detection refers tomeasurement of a physical phenomenon, such as energy or particleemission or absorption, of an atom, molecule or composition that bindsdirectly or indirectly to the detectable moiety (e.g., detection of alabeled secondary antibody or antigen-binding fragment thereof thatbinds to a primary antibody (e.g., an anti-RSV antibody orantigen-binding fragment thereof provided herein).

As used herein, the term “subject” refers to an animal, including amammal, such as a human being.

As used herein, a patient refers to a human subject.

As used herein, animal includes any animal, such as, but are not limitedto primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer, sheep; pigs and other animals. Non-human animals exclude humans asthe contemplated animal. The polypeptides provided herein are from anysource, animal, plant, prokaryotic and fungal. Most polypeptides are ofanimal origin, including mammalian origin.

As used herein, a “elderly,” refers to refers to a subject, who due toage has a decreased immune response and has a decreased response tovaccination. Typically, an elderly subject is one that is human that issixty-five and greater years of age, more typically, 70 and greateryears of age.

As used herein, a “human infant” refers to a human less than or about 24months (e.g., less than or about 16 months, less than or about 12months, less than or about 6 months, less than or about 3 months, lessthan or about 2 months, or less than or about 1 month of age).Typically, the human infant is born at more than 38 weeks of gestationalage.

As used herein, a “human infant born prematurely” refers to a human bornat less than or about 40 weeks gestational age, typically, less than orabout 38 weeks gestational age.

As used herein, a “unit dose form” refers to physically discrete unitssuitable for human and animal subjects and packaged individually as isknown in the art.

As used herein, a “single dosage formulation” refers to a formulationfor direct administration.

As used herein, an “article of manufacture” is a product that is madeand sold. As used throughout this application, the term is intended toencompass any of the compositions provided herein contained in articlesof packaging.

As used herein, a “fluid” refers to any composition that can flow.Fluids thus encompass compositions that are in the form of semi-solids,pastes, solutions, aqueous mixtures, gels, lotions, creams and othersuch compositions.

As used herein, an isolated or purified polypeptide or protein (e.g. anisolated antibody or antigen-binding fragment thereof) orbiologically-active portion thereof (e.g. an isolated antigen-bindingfragment) is substantially free of cellular material or othercontaminating proteins from the cell or tissue from which the protein isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. Preparations can be determined tobe substantially free if they appear free of readily detectableimpurities as determined by standard methods of analysis, such as thinlayer chromatography (TLC), gel electrophoresis and high performanceliquid chromatography (HPLC), used by those of skill in the art toassess such purity, or sufficiently pure such that further purificationdoes not detectably alter the physical and chemical properties, such asenzymatic and biological activities, of the substance. Methods forpurification of the compounds to produce substantially chemically purecompounds are known to those of skill in the art. A substantiallychemically pure compound, however, can be a mixture of stereoisomers. Insuch instances, further purification might increase the specificactivity of the compound. As used herein, a “cellular extract” or“lysate” refers to a preparation or fraction which is made from a lysedor disrupted cell.

As used herein, isolated nucleic acid molecule is one which is separatedfrom other nucleic acid molecules which are present in the naturalsource of the nucleic acid molecule. An “isolated” nucleic acidmolecule, such as a cDNA molecule, can be substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Exemplary isolated nucleic acidmolecules provided herein include isolated nucleic acid moleculesencoding an antibody or antigen-binding fragments provided.

As used herein, a “control” refers to a sample that is substantiallyidentical to the test sample, except that it is not treated with a testparameter, or, if it is a plasma sample, it can be from a normalvolunteer not affected with the condition of interest. A control alsocan be an internal control.

As used herein, a “composition” refers to any mixture. It can be asolution, suspension, liquid, powder, paste, aqueous, non-aqueous or anycombination thereof.

As used herein, a “combination” refers to any association between oramong two or more items. The combination can be two or more separateitems, such as two compositions or two collections, can be a mixturethereof, such as a single mixture of the two or more items, or anyvariation thereof. The elements of a combination are generallyfunctionally associated or related.

As used herein, combination therapy refers to administration of two ormore different therapeutics, such as two or more different anti-RSVantibodies and/or anti-RSV antibodies and antigen-binding fragmentsthereof. The different therapeutic agents can be provided andadministered separately, sequentially, intermittently, or can beprovided in a single composition.

As used herein, a kit is a packaged combination that optionally includesother elements, such as additional reagents and instructions for use ofthe combination or elements thereof, for a purpose including, but notlimited to, activation, administration, diagnosis, and assessment of abiological activity or property.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a polypeptide, comprising “an immunoglobulindomain” includes polypeptides with one or a plurality of immunoglobulindomains.

As used herein, the term “or” is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 amino acids” means “about 5 amino acids” and also “5 aminoacids.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally variantportion means that the portion is variant or non-variant.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, Biochem. (1972)11(9):1726-1732).

B. OVERVIEW

Provided are anti-RSV antibodies or antigen-binding fragments thereofthat bind to and neutralize respiratory syncytial virus. The anti-RSVantibodies provided herein are neutralizing antibodies that recognizeone or more epitopes on the surface of RSV. In particular, theantibodies provided herein bind to a RSV fusion (F) protein. Theantibodies provided herein can be used in prophylaxis therapies. Theantibodies provided herein also can be used as therapeutics.

For example, the antibodies provided can be employed for the preventionand/or spread of pathogenic disease, including, but not limited to theinhibition of viral transmission between subjects, inhibition ofestablishment of viral infection in a host, and reduction of viral loadin a subject. The antibodies also can be employed for preventing,treating, and/or alleviating one or more symptoms of a RSV infection orfor reducing the duration of a RSV infection. Accordingly, treatment ofpatients with antibodies provided herein can decrease the mortalityand/or morbidity rate associated with RSV infection.

RSV persistence is associated with the generation of escape mutants thatcannot be neutralized by an antibody. Thus, the main challenges todevelopment of therapeutic anti-viral antibodies are the generation oridentification of antibodies that have a neutralization epitope thatis 1) conserved across various strains or serotypes and 2) is difficultfor the evolving virus to generate escape mutants against. Antibodiesprovided herein bind to various RSV subgroups and strains. Antibodiesprovided herein also exhibit improved virus neutralization activitycompared to existing antibodies in the prior art. The providedantibodies effectively neutralize virus over successive rounds ofreplication, where RSV typically would generate escape mutants to resistneutralization. The ability to limit the generation of MARMs means thatthe antibodies provided herein bind to an epitope that is lesssusceptible to variation in the form of generated escape mutants. Thisepitope, therefore, is different from epitopes of other known anti-RSVantibodies. Thus, the provided anti-RSV antibodies, in addition toprophylaxis therapy, also are useful for the treatment of RSV infection.Currently, there are no known approved antibody therapeutics against RSVinfection. As such, the antibodies provided herein are especiallyimportant for treatment of RSV infection among elderly patients, forexample those in group or retirement homes, where proximity increasesthe risk for viral spread among patients. Treatment with the antibodiesprovided herein is also important in situations where non-compliancewith dosage regimes increases risk for viral escape, as non-compliancein the prophylaxis treatment of RSV with palivizumab is increasinglyleading causing viral resistance (see, e.g., Adams et al., (2010) ClinInfect Dis. 51(2):185-188).

Generally, the anti-RSV antibodies provided herein bind to RSV F proteinwith high affinity. Compared to existing approved anti-RSV antibodies(e.g. palivizumab; SYNAGIS®), the high affinity anti-RSV antibodiesprovided herein allow for less frequent administration for preventingand/or treating a RSV infection, for preventing, treating, and/oralleviating one or more symptoms of a RSV infection, or for reducing theduration of a RSV infection. Thus, the anti-RSV antibodies providedherein are useful as therapeutic antibodies, i.e., for treatment of RSVinfection. Less frequent administration allows easier compliance withdosing regimes and therefore lessens the possibility of missed dosageswhich lead to increased viral resistance to the anti-RSV antibody. Lowerdoses of antibodies that immunospecifically bind to RSV also can reducethe likelihood of adverse effects of immunoglobulin therapy.

Generally, the anti-RSV antibodies provided herein have the ability toinhibit or reduce one or more activities of the virus, such as, forexample, association of the virus with a target cell membrane, fusion ofthe virus with the target cell membrane and/or cell entry, production ofnew viral particles, including inhibition of viral replication, or cellto cell fusion of an infected cell with another cell (i.e. syncytiaformation). The provided anti-RSV antibodies also can be employed toincrease the immune the response against a RSV infection.

1. Respiratory Syncytial Virus

Human RSV is a member of the Pneumovirus subfamily of the familyParamyxoviridae. There are two distinct subgroups of human RSV, group Aand group B. Additionally, each subtype is further divided into twostrains, A1 and A2, and B1 and B2. RSV is an enveloped, non-segmented,negative-sense RNA virus with a genome of composed of approximately15,000 nucleotides that encode eleven viral proteins.

RSV encodes two major surface glycoproteins, glycoprotein G andglycoprotein F. Glycoprotein G, or the attachment protein, mediatesvirus binding to the cell receptor while glycoprotein F, or the fusionprotein, promotes fusion of the viral and cell membranes, allowingpenetration of the viral ribonucleoprotein into the cell cytoplasm(Lopez et al. (1998) J. Virology 72:6922-6928). Glycoprotein F alsopromotes fusion of the membranes of infected cells with those ofadjacent cells leading to the formation of syncytia. The F proteincontains two disulfide-linked subunits, F₁ and F₂, which are produced byproteolytic cleavage of an inactive, N-glycosylated precursor. The Gprotein is a 80-90 kDa type II transmembrane glycoprotein, containing N-and O-linked oligosaccharides attached to a 32 kDa precursor protein.

Antibodies prepared against RSV F or G glycoproteins have been shown toneutralize RSV with high efficiency in vitro and have prophylacticeffects in vivo (see e.g., Walsh et al. (1986) J. Gen. Microbiol.67:505; Beeler et al. (1989) J. Virol. 63:2941-2950, Garcia-Borreno etal. (1989) J. Virol. 63:925-932, Taylor et al. (1984) Immunology52:137-142, and U.S. Pat. Nos. 5,824,307 and 6,818,216). Antibodiesdirected against RSV F protein also are effective in inhibiting fusionof RSV-infected cells with neighboring uninfected cells.

Analysis of various monoclonal antibodies that immunospecifically bindto the RSV F protein have led to the identification of threenon-overlapping antigenic sites, A, B, and C and one bridge site. AB(Beeler et al. (1989) J. Virol. 63:2941-2950). Each of the antigenicsites contain distinct epitopes. In one study of a panel of 18monoclonal antibodies, five epitopes of antigenic site A, four epitopesof antigenic site B, and four epitopes of antigenic site C wereidentified based on monoclonal antibody escape mutants (MARMs) (see,e.g., Beeler et al. (1989) J. Virol. 63:2941-2950). Monoclonal antibody1129, which binds to antigenic site A epitope 4 (Beeler et al. (1989) J.Virology 63(7):2841-2950), is the parental antibody from which thehumanized palivizumab (SYNAGIS®) was generated (see Johnson et al.(1997) J. Infect. Diseases 176:1215-1224 and U.S. Pat. No. 5,824,307).Additional RSV F protein epitopes also have been identified. Forexample, the human anti-RSV Fab fragment Fab 19 (see Barbas et al.(1992) Proc. Natl. Acad Sci. USA 89:10164-10168 and Crowe et al., (1994)Proc. Natl. Acad Sci USA 91:1386-1390) binds to an epitope in antigenicsite A that differs from the epitopes identified by Beeler et al. (seeCrowe et al. (1998) Virology 252:373-375) and Barbas et al. (1992) Proc.Natl. Acad. Sci. USA 89:10164-10168).

The RSV F protein exhibits over 91% similarity across the RSV A and Bsubgroups, while the RSV G protein exhibits only 53% amino acidsimilarity between the RSV A and RSV B subgroups (Sullender (2000) Clin.Microbial Rev. 13:1-15). Because A and B virus subtypes co-circulate inmost RSV epidemics, an antibody that neutralizes A and B subtypes ofRSV, such as the anti-RSV antibodies or antigen-binding fragmentsprovided herein, is desirable.

Human RSV, like most RNA viruses, has the capacity of undergoing rapidmutations under selective pressure. The selection of RSV escape mutants(MARMs) in vitro using monoclonal antibodies is well documented (e.g.Garcia-Barreno et al. (1989) J. Virol. 63:925-932). For example, it hasbeen shown that single amino acid mutations at amino acid residues N262,I266, N268, K272, S275, N276, P389 or R429, or double amino acidmutations at F32 and K272 or A241 and K421 in the RSV A2 F proteineffect escape of known anti-RSV monoclonal antibodies (see, e.g., Croweet al. (1998) Virology 252:373-375; Zhao et al., (2004) J. InfectiousDisease 190:1941-1946 and Liu et al., (2007) Virology Journal 4:71).Additionally, palivizumab (SYNAGIS®) has been shown to select for escapemutants, in vitro as well as in vivo, and that some of the isolatedmutants are completely resistant to palivizumab prophylaxis in cottonrats (see e.g., Zhao et al. (2005) J. Virol. 79:3962-3968 and Zhao etal. (2004) J. Infect. Dis. 190:1941-1946). For example, it has beendemonstrated that a single amino acid mutation in the RSV F proteinresults in escape from palivizumab (see, Zhao et al., (2004) J.Infectious Disease 190:1941-1946). Thus, an antibody that effectivelyneutralizes the RSV virus over successive rounds of replication withoutgeneration of an escape mutant, such as the anti-RSV antibodies orantigen-binding fragments provided herein, is preferred.

RSV infection is a major cause of lower respiratory tract disease ininfants and small children. RSV infection also is the most common causeof bronchiolitis, or inflammation of the small airways in the lung, andpneumonia in children under 1 year of age in the United States. Inaddition, RSV infection also is recognized as an important cause ofrespiratory illness in older adults. Symptoms and conditions associatedwith RSV infection include, for example, asthma, wheezing, reactiveairway disease (RAD), and chronic obstructive pulmonary disease (COPD).Accordingly, as described herein, the anti-RSV antibodies providedherein can be employed for the prophylaxis and treatment of RSVinfection and/or alleviation of one or more symptoms of suchRSV-mediated diseases.

C. ANTI-RSV ANTIBODIES

Provided herein are anti-RSV antibodies or antigen-binding fragmentsthereof that can be employed for therapeutic, prophylactic anddiagnostic use. The anti-RSV antibodies or antigen-binding fragmentsthereof provided herein can be used, for example, for passiveimmunization of a subject against RSV or for treatment of a subject witha viral infection. In one example, the anti-RSV antibodies orantigen-binding fragments thereof provided herein are used forprophylaxis, i.e., the prevention of RSV infection. In another example,the anti-RSV antibodies or antigen-binding fragments thereof providedherein are used as therapeutic antibodies, i.e., for treatment of a RSVviral infection. In yet another example, the anti-RSV antibodies orantigen-binding fragments thereof provided herein are used for passiveimmunization of a subject against RSV. The provided anti-RSV antibodiesor antigen-binding fragments thereof also can be used for detection of aRSV infection or for monitoring RSV infection in vitro and in vivo.

1. General Antibody Structure and Functional Domains

Antibodies are produced naturally by B cells in membrane-bound andsecreted forms. Antibodies specifically recognize and bind antigenepitopes through cognate interactions. Antibody binding to cognateantigens can initiate multiple effector functions, which causeneutralization and clearance of toxins, pathogens and other infectiousagents.

Diversity in antibody specificity arises naturally due to recombinationevents during B cell development. Through these events, variouscombinations of multiple antibody V, D and J gene segments, which encodevariable regions of antibody molecules, are joined with constant regiongenes to generate a natural antibody repertoire with large numbers ofdiverse antibodies. A human antibody repertoire contains more than 10¹⁰different antigen specificities and thus theoretically can specificallyrecognize any foreign antigen. Antibodies include such naturallyproduced antibodies, as well as synthetically, i.e. recombinantly,produced antibodies, such as antibody fragments, including the anti-RSVantibodies or antigen-binding fragments thereof provided herein.

In folded antibody polypeptides, binding specificity is conferred byantigen-binding site domains, which contain portions of heavy and/orlight chain variable region domains. Other domains on the antibodymolecule serve effector functions by participating in events such assignal transduction and interaction with other cells, polypeptides andbiomolecules. These effector functions cause neutralization and/orclearance of the infecting agent recognized by the antibody. Domains ofantibody polypeptides can be varied according to the methods herein toalter specific properties.

a. Structural and Functional Domains of Antibodies

Full-length antibodies contain multiple chains, domains and regions. Afull length conventional antibody contains two heavy chains and twolight chains, each of which contains a plurality of immunoglobulin (Ig)domains. An Ig domain is characterized by a structure called the Igfold, which contains two beta-pleated sheets, each containinganti-parallel beta strands connected by loops. The two beta sheets inthe Ig fold are sandwiched together by hydrophobic interactions and aconserved intra-chain disulfide bond. The Ig domains in the antibodychains are variable (V) and constant (C) region domains. Each heavychain is linked to a light chain by a disulfide bond, and the two heavychains are linked to each other by disulfide bonds. Linkage of the heavychains is mediated by a flexible region of the heavy chain, known as thehinge region.

Each full-length conventional antibody light chain contains one variableregion domain (V_(L)) and one constant region domain (C_(L)). Eachfull-length conventional heavy chain contains one variable region domain(V_(H)) and three or four constant region domains (C_(H)) and, in somecases, hinge region. Owing to recombination events discussed above,nucleic acid sequences encoding the variable region domains differ amongantibodies and confer antigen-specificity to a particular antibody. Theconstant regions, on the other hand, are encoded by sequences that aremore conserved among antibodies. These domains confer functionalproperties to antibodies, for example, the ability to interact withcells of the immune system and serum proteins in order to causeclearance of infectious agents. Different classes of antibodies, forexample IgM, IgD, IgG, IgE and IgA, have different constant regions,allowing them to serve distinct effector functions.

Each variable region domain contains three portions calledcomplementarity determining regions (CDRs) or hypervariable (HV)regions, which are encoded by highly variable nucleic acid sequences.The CDRs are located within the loops connecting the beta sheets of thevariable region Ig domain. Together, the three heavy chain CDRs (CDR1,CDR2 and CDR3) and three light chain CDRs (CDR1, CDR2 and CDR3) make upa conventional antigen-binding site (antibody combining site) of theantibody, which physically interacts with cognate antigen and providesthe specificity of the antibody. A whole antibody contains two identicalantibody combining sites, each made up of CDRs from one heavy and onelight chain. Because they are contained within the loops connecting thebeta strands, the three CDRs are non-contiguous along the linear aminoacid sequence of the variable region. Upon folding of the antibodypolypeptide, the CDR loops are in close proximity, making up the antigencombining site. The beta sheets of the variable region domains form theframework regions (FRs), which contain more conserved sequences that areimportant for other properties of the antibody, for example, stability.

b. Antibody Fragments

Antibodies provided herein include antibody fragments, which arederivatives of full-length antibody that contain less than the fullsequence of the full-length antibodies but retain at least a portionspecific binding abilities of the full-length antibody. The antibodyfragments also can include antigen-binding portions of an antibody thatcan be inserted into an antibody framework (e.g., chimeric antibodies)in order to retain the binding affinity of the parent antibody. Examplesof antibody fragments include, but are not limited to, Fab, Fab′,F(ab′)₂, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd′fragments, and other fragments, including modified fragments (see, forexample, Methods in Molecular Biology, Vol 207: Recombinant Antibodiesfor Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25,Kipriyanov). Antibody fragments can include multiple chains linkedtogether, such as by disulfide bridges and can be producedrecombinantly. Antibody fragments also can contain synthetic linkers,such as peptide linkers, to link two or more domains. Methods forgenerating antigen-binding fragments are well-known known in the art andcan be used to modify any antibody provided herein. Fragments ofantibody molecules can be generated, such as for example, by enzymaticcleavage. For example, upon protease cleavage by papain, a dimer of theheavy chain constant regions, the Fc domain, is cleaved from the two Fabregions (i.e. the portions containing the variable regions).

Single chain antibodies can be recombinantly engineered by joining aheavy chain variable region (V_(H)) and light chain variable region(V_(L)) of a specific antibody. The particular nucleic acid sequencesfor the variable regions can be cloned by standard molecular biologymethods, such as, for example, by polymerase chain reaction (PCR) andother recombination nucleic acid technologies. Methods for producingsFvs are described, for example, by Whitlow and Filpula (1991) Methods,2: 97-105; Bird et al. (1988) Science 242:423-426: Pack et al. (1993)Bio/Technology 11:1271-77; and U.S. Pat. Nos. 4,946,778, 5,840,300,5,667,988, 5,658,727, 5,258,498). Single chain antibodies also can beidentified by screening single chain antibody libraries for binding to atarget antigen. Methods for the construction and screening of suchlibraries are well-known in the art.

2. Exemplary Anti-RSV Antibodies

Provided herein are antibodies or antigen-binding fragments thereof thatbind to and neutralize RSV. In particular the antibodies orantigen-binding fragments immunospecifically bind to a RSV F protein.For example, the anti-RSV antibodies provided herein immunospecificallybind to an isolated or purified or recombinant RSV F protein. In otherexamples, the anti-RSV antibodies provided herein immunospecificallybind to the RSV virion or RSV virus encoding surface glycoprotein F (RSVF protein).

The anti-RSV antibodies provided herein exhibit properties that areadvantageous or different from anti-RSV antibodies in the art. Forexample, antibodies provided herein include anti-RSV antibodies thatexhibit lower or improved binding affinity for RSV F protein, such anlower or improved binding affinity for an isolated or purified orrecombinant RSV F protein or for the RSV virus or viron expressingnative F protein. In other examples, antibodies provided herein includeanti-RSV antibodies that exhibit an improved or better neutralizationactivity of RSV. In particular examples, anti-RSV antibodies providedherein include antibodies that bind to an epitope on RSV that is lesssusceptible to variation in the form of generated escape mutants (MARMs)compared to other prior art or existing anti-RSV antibodies. Forexample, in the presence of an anti-RSV antibody provided herein, forexample the antibody 30D8 or a full-length or other antibody fragmentform thereof, RSV is incapable of generating an escape mutant after morethan 10 rounds of viral replication, and generally after more than 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 rounds.

Anti-RSV antibodies include the antibodies sc5 or 58c5, or full-lengthor other antibody fragment forms thereof (see e.g. U.S. Patentpublication No. US2011/0076268 and International Published PCTApplication No. WO2011/020079). 58c5 is a Fab fragment that contains aheavy chain having the amino sequence set forth in SEQ ID NO:1 and alight chain having the amino acid sequence set forth in SEQ ID NO:5.Antibody or antigen-fragment forms of 58c5 and that bind to the sameepitope as 58c5 contain a heavy chain having complementarity determiningregions (CDRs) V_(H) CDR1 set forth as GASINSDNYYWT (SEQ ID NO:2) or SEQID NO:435, a V_(H) CDR2 set forth as HISYTGNTYYTPSLKS (SEQ ID NO:3), anda V_(H) CDR3 set forth as CGAYVLISNCGWFDS (SEQ ID NO:4; and a lightchain having CDRs V_(L) CDR1 set forth as QASQDISTYLN (SEQ ID NO:6), aV_(L) CDR2 set forth as GASNLET (SEQ ID NO:7), and a V_(L) CDR3 setforth as QQYQYLPYT (SEQ ID NO:8). Generally, antibody fragment form of58c5 include the VH domain having the amino acid sequence set forth inamino acids 1-125 of SEQ ID NO:1 and a VL domain having the amino acidsequence set forth in amino acids 1-107 of SEQ ID NO:5. sc5 is a Fabfragment that contains a heavy chain having the amino acid sequence setforth in SEQ ID NO:9 and a light chain having the amino acid sequenceset forth in SEQ ID NO:13. Antibody or antigen-fragment forms of sc5 andthat bind to the same epitope as sc5 contain a heavy chain havingcomplementarity determining regions (CDRs) V_(H) CDR1 set forth asGDSISGSNWWN (SEQ ID NO:10) or SEQ ID NO:436, a V_(H) CDR2 set forth asEIYYRGTTNYKSSLKG (SEQ ID NO:11), and a V_(H) CDR3 set forth asGGRSTFGPDYYYYMDV (SEQ ID NO:12); and a light chain having CDRs V_(L)CDR1 set forth as RASQNIKNYLN (SEQ ID NO:14), a V_(L) CDR2 set forth asAASTLQS (SEQ ID NO:15), and a V_(L) CDR3 set forth as QQSYNNQLT (SEQ IDNO:16). Generally, antibody fragment form of sc5 includes the VH domainhaving the amino acid sequence set forth in amino acids 1-125 of SEQ IDNO:9 and a VL domain having the amino acid sequence set forth in aminoacids 1-107 of SEQ ID NO:13.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein include monoclonal antibodies, multispecific antibodies,bispecific antibodies, human antibodies, humanized antibodies, camelisedantibodies, chimeric antibodies, single-chain Fvs (scFv), single chainantibodies, single domain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies,intrabodies, or antigen-binding fragments of any of the above. Inparticular, antibodies include immunoglobulin molecules andimmunologically active fragments of immunoglobulin molecules, i.e.,molecules that contain an antigen-binding site. Immunoglobulin moleculescan be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be used in the methods of treatment and diagnosis in formsthat include monoclonal antibodies, multispecific antibodies, humanantibodies, humanized antibodies, camelised antibodies, chimericantibodies, single-chain Fvs (scFv), single chain antibodies, singledomain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs(sdFv), and anti-idiotypic (anti-Id) antibodies, intrabodies, orantigen-binding fragments of any of the above. In particular, theantibodies include immunoglobulin molecules and immunologically activefragments of immunoglobulin molecules, i.e., molecules that contain anantigen-binding site. Immunoglobulin molecules can be of any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) or subclass.

Exemplary anti-RSV antibodies or antigen-binding fragments thereofprovided herein that immunospecifically bind to a RSV F protein include30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 and 69F6, which are Fabfragments described in detail elsewhere herein. Exemplary anti-RSVantibodies or antigen-binding fragments thereof provided herein alsoinclude anti-RSV antibodies or antigen-binding fragments thereof thatcontain a heavy chain, which contains a variable heavy (V_(H)) domainand a constant heavy domain 1 (C_(H)1) and/or a light chain, whichcontains a variable light (V_(L)) domain and a constant light domain(C_(L)) of 30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 or 69F6. Forexample, exemplary anti-RSV antibodies or antigen-binding fragmentsthereof provided herein include anti-RSV antibodies or antigen-bindingfragments thereof that contain a heavy chain having the amino acidsequence set forth in SEQ ID NO:396, 398, 400, 402, 404, 452, 454 or 456and/or a light chain having the amino acid sequence set forth in SEQ IDNO:395, 397, 399, 401, 403, 453, 455 or 457. In a particular example,the anti-RSV antibody is a Fab fragment that contains a heavy chainhaving the amino acid sequence set forth in SEQ ID NO:396 and a lightchain having the amino acid sequence set forth in SEQ ID NO:395. In aparticular example, the anti-RSV antibody is a Fab fragment thatcontains a heavy chain having the amino acid sequence set forth in SEQID NO:398 and a light chain having the amino acid sequence set forth inSEQ ID NO:397. In a particular example, the anti-RSV antibody is a Fabfragment that contains a heavy chain having the amino acid sequence setforth in SEQ ID NO:400 and a light chain having the amino acid sequenceset forth in SEQ ID NO:399. In a particular example, the anti-RSVantibody is a Fab fragment that contains a heavy chain having the aminoacid sequence set forth in SEQ ID NO:402 and a light chain having theamino acid sequence set forth in SEQ ID NO:401. In a particular example,the anti-RSV antibody is a Fab fragment that contains a heavy chainhaving the amino acid sequence set forth in SEQ ID NO:404 and a lightchain having the amino acid sequence set forth in SEQ ID NO:403. In aparticular example, the anti-RSV antibody is a Fab fragment thatcontains a heavy chain having the amino acid sequence set forth in SEQID NO:452 and a light chain having the amino acid sequence set forth inSEQ ID NO:453. In a particular example, the anti-RSV antibody is a Fabfragment that contains a heavy chain having the amino acid sequence setforth in SEQ ID NO:454 and a light chain having the amino acid sequenceset forth in SEQ ID NO:455. In a particular example, the anti-RSVantibody is a Fab fragment that contains a heavy chain having the aminoacid sequence set forth in SEQ ID NO:456 and a light chain having theamino acid sequence set forth in SEQ ID NO:457.

The antibodies provided herein include full-length antibody forms of30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 or 69F6. The antibodiesprovided herein also include full-length antibody forms containing theantigen-binding site (e.g. CDRs) of 30D8, 104E5, 38F10, 14G3, 90D3,56E11, 17C9 or 69F6. Full-length anti-RSV antibodies or antigen-bindingfragments thereof provided herein can contain any light chain and heavychain constant region known in the art, such as any human constantregion known in the art. For example, full-length antibodies providedherein can contain a light chain constant region (CL) that is a humanlight chain kappa (κ), human light chain lambda (λ). Full-lengthantibodies provided herein can contain a heavy chain constant region(C_(H)1-linker-C_(H)2-C_(H)3) that is of any isotype, and in particularthat is an IgG. The constant region can be the constant region ofsubclass IgG1 (SEQ ID NO:356), the constant region of IgG2 (SEQ IDNO:357), the constant region of IgG3 (SEQ ID NO:358) or the constantregion of IgG4 (SEQ ID NO:359). In particular, full-length antibodyforms of any of the antibodies provided herein contain the constantregion of IgG, subclass IgG1.

The antibodies provided herein include other antibody fragment forms of30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 and 69F6 thatimmunospecifically bind an RSV F protein. Such fragments include anyantigen-binding fragment thereof or an engineered antibody containing anantigen-binding fragment(s) of 30D8, 104E5, 38F10, 14G3, 90D3, 56E11,17C9 or 69F6 that retains the ability to bind an RSV F protein. Suchantibodies include, for example, chimeric antibodies, single-chain Fvs(scFv), single chain antibodies, single domain antibodies, Fabfragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), anti-idiotypic(anti-Id) antibodies, intrabodies, or antigen-binding fragments of anyof the above. In particular examples, the antibody is the Fab 30D8,104E5, 38F10, 14G3, 90D3, 56E11, 17C9 or 69F6.

Exemplary anti-RSV antibodies or antigen-binding fragments thereofprovided herein include anti-RSV antibodies or antigen-binding fragmentsthereof that contain a V_(H) domain and/or a variable light V_(L) domainhaving an amino acid sequence of the V_(H) domain and/or V_(L) domain,respectively, of 30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 or 69F6.For example, an antibody or antigen-binding fragment thereof can containa V_(H) domain having the amino acid sequence set forth in amino acids1-125 of SEQ ID NO:398 or 402, amino acids 1-121 of SEQ ID NO:396, aminoacids 1-123 of SEQ ID NO:404, amino acids 1-124 of SEQ ID NO:400 or 452,amino acids 1-133 of SEQ ID NO:454 or amino acids 1-118 of SEQ ID NO:456and/or a V_(L) domain having the amino acid sequence set forth in aminoacids 1-107 of SEQ ID NO: 397, 403 or 455, amino acids 1-108 of SEQ IDNO:399, amino acids 1-110 of SEQ ID NO:395, amino acids 1-113 of SEQ IDNO:401, amino acids 1-111 of SEQ ID NO:453 or amino acids 1-109 of SEQID NO:457.

Exemplary anti-RSV antibodies or antigen-binding fragments thereofprovided herein include anti-RSV antibodies or antigen-binding fragmentsthereof that contain a V_(H) domain and/or a V_(L) domain having anamino acid sequence that is at least or about 80% identical to the V_(H)domain and/or V_(L) domain, respectively, of 30D8, 104E5, 38F10, 14G3,90D3, 56E11, 17C9 or 69F6. For example, the antibody or antigen-bindingfragment thereof provided herein can contain a V_(H) domain having theamino acid sequence that is 80% identical to the amino acid sequence setforth in amino acids 1-125 of SEQ ID NO:398 or 402, amino acids 1-121 ofSEQ ID NO:396, amino acids 1-123 of SEQ ID NO:404, amino acids 1-124 ofSEQ ID NO:400 or 452, amino acids 1-133 of SEQ ID NO:454 or amino acids1-118 of SEQ ID NO:456 and/or a V_(L) domain having the amino acidsequence set forth in amino acids 1-107 of SEQ ID NO:397, 403 or 455,amino acids 1-108 of SEQ ID NO:399, amino acids 1-110 of SEQ ID NO:395,amino acids 1-113 of SEQ ID NO:401, amino acids 1-111 of SEQ ID NO:453or amino acids 1-109 of SEQ ID NO:457.

In some examples, the anti-RSV antibody or antigen-binding fragmentthereof provided herein can contain a V_(H) domain having the amino acidsequence that is at least or about 81%, at least or about 82%, at leastor about 83%, at least or about 84%, at least or about 85%, at least orabout 86%, at least or about 87%, at least or about 88%, at least orabout 89%, at least or about 90%, at least or about 91%, at least orabout 92%, at least or about 93%, at least or about 94%, at least orabout 95%, at least or about 96%, at least or about 97%, at least orabout 98%, or at least or about 99% identical to the amino acid sequenceset forth in amino acids 1-125 of SEQ ID NO:398 or 402, amino acids1-121 of SEQ ID NO:396, amino acids 1-123 of SEQ ID NO:404, amino acids1-124 of SEQ ID NO:400 or 452, amino acids 1-133 of SEQ ID NO:454 oramino acids 1-118 of SEQ ID NO:456 and/or a V_(L) domain having theamino acid sequence that is at least or about 81%, at least or about82%, at least or about 83%, at least or about 84%, at least or about85%, at least or about 86%, at least or about 87%, at least or about88%, at least or about 89%, at least or about 90%, at least or about91%, at least or about 92%, at least or about 93%, at least or about94%, at least or about 95%, at least or about 96%, at least or about97%, at least or about 98%, or at least or about 99% identical to theamino acid sequence set forth in amino acids 1-107 of SEQ ID NO:397, 403or 455, amino acids 1-108 of SEQ ID NO:399, amino acids 1-110 of SEQ IDNO:395, amino acids 1-113 of SEQ ID NO:401, amino acids 1-111 of SEQ IDNO:453 or amino acids 1-109 of SEQ ID NO:457.

Thus, provided herein is an antibody or antigen-binding fragment thereofthat contains a V_(H) domain having an amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-121 of SEQ ID NO:396 andthat contains a V_(L) domain having the amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-110 of SEQ ID NO:395.

Thus, provided herein is an antibody or antigen-binding fragment thereofthat contains a V_(H) domain having an amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-125 of SEQ ID NO:398 andthat contains a V_(L) domain having the amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-107 of SEQ ID NO:397.

Thus, provided herein is an antibody or antigen-binding fragment thereofthat contains a V_(H) domain having an amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-124 of SEQ ID NO:400 andthat contains a V_(L) domain having the amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-108 of SEQ ID NO:399.

Thus, provided herein is an antibody or antigen-binding fragment thereofthat contains a V_(H) domain having an amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-125 of SEQ ID NO:402 andthat contains a V_(L) domain having the amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-113 of SEQ ID NO:401.

Thus, provided herein is an antibody or antigen-binding fragment thereofthat contains a V_(H) domain having an amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-123 of SEQ ID NO:404 andthat contains a V_(L) domain having the amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-107 of SEQ ID NO:403.

Thus, provided herein is an antibody or antigen-binding fragment thereofthat contains a V_(H) domain having an amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-124 of SEQ ID NO:452 andthat contains a V_(L) domain having the amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-111 of SEQ ID NO:453.

Thus, provided herein is an antibody or antigen-binding fragment thereofthat contains a V_(H) domain having an amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-133 of SEQ ID NO:454 andthat contains a V_(L) domain having the amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-107 of SEQ ID NO:455.

Thus, provided herein is an antibody or antigen-binding fragment thereofthat contains a V_(H) domain having an amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-118 of SEQ ID NO:456 andthat contains a V_(L) domain having the amino acid sequence that is atleast or that is about 80% to 99% identical, for example, 90% to 99% orat least 95% identical, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence set forth in amino acids 1-109 of SEQ ID NO:457.

Also provided are anti-RSV antibodies or antigen-binding fragmentsthereof that contain one or more V_(H) complementarity determiningregions (CDRs) selected from among the CDRs of 30D8, 104E5, 38F10, 14G3,90D3, 56E11, 17C9 or 69F6. For example, the anti-RSV antibody orantigen-binding fragment thereof can contain a V_(H) CDR1 having theamino acid sequence set forth in SEQ ID NOS:405, 411, 417, 423, 429,437-441, 458, 464, 470 or 482-484. For example, the anti-RSV antibody orantigen-binding fragment thereof can contain a V_(H) CDR1 having theamino acid sequence GFTFSGHTIA (SEQ ID NO:405), GGTFDTYTIS (SEQ IDNO:411), GFSITDFGIH (SEQ ID NO:417), GASISSDNHYWS (SEQ ID NO:423),GFTLKNYEMN (SEQ ID NO:429), GHTIA (SEQ ID NO:437), TYTIS (SEQ IDNO:438), DFGIH (SEQ ID NO:439), SDNHYWS (SEQ ID NO:440), NYEMN (SEQ IDNO:441), GVSINSNNYFWA (SEQ ID NO:458), GDSFNDYFWT (SEQ ID NO:464),GYSFTSYWIA (SEQ ID NO:470), SNNYFWA (SEQ ID NO:482), DYFWT (SEQ IDNO:483) or SYWIA (SEQ ID NO:484).

In another example, the anti-RSV antibody or antigen-binding fragmentthereof can contain a V_(H) CDR2 having the amino acid sequence setforth in SEQ ID NOS:406, 412, 418, 424, 430, 459, 465 or 471. Forexample, the anti-RSV antibody or antigen-binding fragment thereof cancontain a V_(H) CDR2 having the amino acid sequence WVSTNNGNTEYAQKIQG(SEQ ID NO:406), RIIPSLGETNYAHKLQG (SEQ ID NO:412), LISYNEVNIHYGESVRG(SEQ ID NO:418), SIYYTGGTNYNPSLKS (SEQ ID NO:424), YISSSGNVVKYVDSVQG(SEQ ID NO:430). NIYYGGSTHYNASLQS (SEQ ID NO:459), EISHSGSTNYSPSLKS (SEQID NO:465) or IIFPNDSDATYSPSFQG (SEQ ID NO:471).

In another example, the anti-RSV antibody or antigen-binding fragmentthereof can contain a V_(H) CDR3 having the amino acid sequence setforth in SEQ ID NOS:407, 413, 419, 425, 431, 460, 466 or 472. Forexample, the anti-RSV antibody or antigen-binding fragment thereof cancontain a V_(H) CDR3 having the amino acid sequence EWLVMGGFAFDH (SEQ IDNO:407), RITGPVDWVWDYGMDV (SEQ ID NO:413), DVWEDSWLSLACFQE (SEQ IDNO:419), GLFFITARPYWYFDL (SEQ ID NO:425) or GFSIDKYDSSVDEY (SEQ IDNO:431), SESIFWDYYYGLDV (SEQ ID NO:460), GVRSRPPPSYRGSGSPPYYHYGMDV (SEQID NO:466) or QYYLGSFES (SEQ ID NO:472).

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(H) CDR1 having the amino acid sequenceset forth in SEQ ID NO:405, a V_(H) CDR2 having the amino acid sequenceset forth in SEQ ID NO:406, and a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NO:407. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(H)CDR1 having the amino acid sequence set forth in SEQ ID NO:437, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NO:406, and aV_(H) CDR3 having the amino acid sequence set forth in SEQ ID NO:407.

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(H) CDR1 having the amino acid sequenceset forth in SEQ ID NO:411, a V_(H) CDR2 having the amino acid sequenceset forth in SEQ ID NO:412, and a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NO:413. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(H)CDR1 having the amino acid sequence set forth in SEQ ID NO:438, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NO:412, and aV_(H) CDR3 having the amino acid sequence set forth in SEQ ID NO:413.

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(H) CDR1 having the amino acid sequenceset forth in SEQ ID NO:417, a V_(H) CDR2 having the amino acid sequenceset forth in SEQ ID NO:418, and a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NO:419. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(H)CDR1 having the amino acid sequence set forth in SEQ ID NO:439, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NO:418, and aV_(H) CDR3 having the amino acid sequence set forth in SEQ ID NO:419.

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(H) CDR1 having the amino acid sequenceset forth in SEQ ID NO:423, a V_(H) CDR2 having the amino acid sequenceset forth in SEQ ID NO:424, and a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NO:425. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(H)CDR1 having the amino acid sequence set forth in SEQ ID NO:440, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NO:424, and aV_(H) CDR3 having the amino acid sequence set forth in SEQ ID NO:425.

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(H) CDR1 having the amino acid sequenceset forth in SEQ ID NO:429, a V_(H) CDR2 having the amino acid sequenceset forth in SEQ ID N0:430, and a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NO:431. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(H)CDR1 having the amino acid sequence set forth in SEQ ID NO:441, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NO:430, and aV_(H) CDR3 having the amino acid sequence set forth in SEQ ID NO:431.

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(H) CDR1 having the amino acid sequenceset forth in SEQ ID NO:458, a V_(H) CDR2 having the amino acid sequenceset forth in SEQ ID NO:459, and a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NO:460. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(H)CDR1 having the amino acid sequence set forth in SEQ ID NO:482, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NO:459, and aV_(H) CDR3 having the amino acid sequence set forth in SEQ ID NO:460.

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(H) CDR1 having the amino acid sequenceset forth in SEQ ID NO:464, a V_(H) CDR2 having the amino acid sequenceset forth in SEQ ID NO:465, and a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NO:466. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(H)CDR1 having the amino acid sequence set forth in SEQ ID NO:483, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NO:465, and aV_(H) CDR3 having the amino acid sequence set forth in SEQ ID NO:466.

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(H) CDR1 having the amino acid sequenceset forth in SEQ ID NO:470, a V_(H) CDR2 having the amino acid sequenceset forth in SEQ ID NO:471, and a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NO:472. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(H)CDR1 having the amino acid sequence set forth in SEQ ID NO:484, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NO:471, and aV_(H) CDR3 having the amino acid sequence set forth in SEQ ID NO:472.

Also provided are anti-RSV antibodies or antigen-binding fragmentsthereof that contain one or more V_(L) complementarity determiningregions (CDRs) selected from among the CDRs of 30D8, 104E5, 38F10, 14G3,90D3, 56E11, 17C9 or 69F6. For example, the anti-RSV antibody orantigen-binding fragment thereof can contain a V_(L) CDR1 having theamino acid sequence set forth in SEQ ID NOS:408, 414, 420, 426, 432,461, 467 or 473. For example, the anti-RSV antibody or antigen-bindingfragment thereof can contain a V_(L) CDR1 having the amino acid sequenceGANNIGSQNVH (SEQ ID NO:408), RASQNIKTYLN (SEQ ID NO:414), RASQSISNWLA(SEQ ID NO:420), RSSQSLLDSDDGNTYLD (SEQ ID NO:426), RASQSISNFLN (SEQ IDNO:432), TGTSSDVGGYNYVS (SEQ ID NO:461), RASQNINTWLA (SEQ ID NO:467) orQASDISNYLN (SEQ ID NO:473).

In another example, the anti-RSV antibody or antigen-binding fragmentthereof can contain a V_(L) CDR2 having the amino acid sequence setforth in SEQ ID NOS:409, 415, 421, 427, 433, 462, 468 or 474. Forexample, the anti-RSV antibody or antigen-binding fragment thereof cancontain a V_(L) CDR2 having the amino acid sequence DDRDRPS(SEQ IDNO:409), AVSNLQS (SEQ ID NO:415), KASNLED (SEQ ID NO:421), TLSYRAS (SEQID NO:427), AASSLQG (SEQ ID NO:433), EVTKRPS (SEQ ID NO:462),AASFLQS(SEQ ID NO:468) or DASYLDT (SEQ ID NO:474).

In another example, the anti-RSV antibody or antigen-binding fragmentthereof can contain a V_(L) CDR3 having the amino acid sequence setforth in SEQ ID NOS:410, 416, 422, 428, 434, 463, 469 or 475. Forexample, the anti-RSV antibody or antigen-binding fragment thereof cancontain a V_(L) CDR3 having the amino acid sequence QVWDSSRDQAVI (SEQ IDNO:410), QQSFSIPLT (SEQ ID NO:416), QQYNSYSGLS (SEQ ID NO:422),MQRMEFPFT (SEQ ID NO:428), QQTYISLYT (SEQ ID NO:434), SSYAGSRHVV (SEQ IDNO:463), QQANSFPRT (SEQ ID NO:469) or QQYDDLRGGFT (SEQ ID NO:475).

In one particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(L) CDR1 having the amino acid sequenceset forth in SEQ ID NO:408, a V_(L) CDR2 having the amino acid sequenceset forth in SEQ ID NO:409, and a V_(L) CDR3 having the amino acidsequence set forth in SEQ ID NO:410. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(L)CDR1 having the amino acid sequence set forth in SEQ ID NO:414, a V_(L)CDR2 having the amino acid sequence set forth in SEQ ID NO:415, and aV_(L) CDR3 having the amino acid sequence set forth in SEQ ID NO:416. Inanother particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(L) CDR1 having the amino acid sequenceset forth in SEQ ID NO:420, a V_(L) CDR2 having the amino acid sequenceset forth in SEQ ID NO:421, and a V_(L) CDR3 having the amino acidsequence set forth in SEQ ID NO:422.

In another particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(L) CDR1 having the amino acid sequenceset forth in SEQ ID NO:426, a V_(L) CDR2 having the amino acid sequenceset forth in SEQ ID NO:427, and a V_(L) CDR3 having the amino acidsequence set forth in SEQ ID NO:428. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(L)CDR1 having the amino acid sequence set forth in SEQ ID NO:432, a V_(L)CDR2 having the amino acid sequence set forth in SEQ ID NO:433, and aV_(L) CDR3 having the amino acid sequence set forth in SEQ ID NO:434. Inanother particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(L) CDR1 having the amino acid sequenceset forth in SEQ ID NO:461, a V_(L) CDR2 having the amino acid sequenceset forth in SEQ ID NO:462, and a V_(L) CDR3 having the amino acidsequence set forth in SEQ ID NO:463. In another particular example, theanti-RSV antibody or antigen-binding fragment thereof contains a V_(L)CDR1 having the amino acid sequence set forth in SEQ ID NO:467, a V_(L)CDR2 having the amino acid sequence set forth in SEQ ID NO:468, and aV_(L) CDR3 having the amino acid sequence set forth in SEQ ID NO:469. Inanother particular example, the anti-RSV antibody or antigen-bindingfragment thereof contains a V_(L) CDR1 having the amino acid sequenceset forth in SEQ ID NO:473, a V_(L) CDR2 having the amino acid sequenceset forth in SEQ ID NO:474, and a V_(L) CDR3 having the amino acidsequence set forth in SEQ ID NO:475.

Any combination of CDRs provided herein can be selected for thegeneration of an antibody or antigen-binding fragment thereof, providedthat the antibody or antigen-binding fragment retains the ability toimmunospecifically bind to a RSV F protein. The anti-RSV antibodies orantigen-binding fragments thereof can contain an antibody frameworkregion known in the art. Exemplary framework regions include isolatednaturally occurring or consensus framework regions, including humanframework regions (see, e.g., Chothia et al. (1998) J. Mol. Biol. 278:457-479). In some examples, the antibody framework region is a humanantibody framework region. In some examples, the antibody orantigen-binding fragment contains a framework region of 30D8, 104E5,38F10, 14G3, 90D3, 56E11, 17C9 or 69F6.

Exemplary isolated anti-RSV antibodies or antigen-binding fragmentsthereof provided herein include any anti-RSV antibody or antigen-bindingfragments thereof that immunospecifically binds to the same epitope on aRSV fusion (F) protein as any of the antibodies provided herein. In oneexample, provided herein is an antibody that binds to the same epitopeas 30D8, which is the antibody that contains a heavy chain set forth inSEQ ID NO:396 and a light chain set forth in SEQ ID NO:395. In anotherexample, provided herein is an antibody that binds to the same epitopeas 104E5, which is the antibody that contains a heavy chain set forth inSEQ ID NO:398 and a light chain set forth in SEQ ID NO:397. In anotherexample, provided herein is an antibody that binds to the same epitopeas 38F10, which is the antibody that contains a heavy chain set forth inSEQ ID NO:400 and a light chain set forth in SEQ ID NO:399. In anotherexample, provided herein is an antibody that binds to the same epitopeas 14G3, which is the antibody that contains a heavy chain set forth inSEQ ID NO:402 and a light chain set forth in SEQ ID NO:401. In anotherexample, provided herein is an antibody that binds to the same epitopeas 90D3, which is the antibody that contains a heavy chain set forth inSEQ ID NO:404 and a light chain set forth in SEQ ID NO:403. In anotherexample, provided herein is an antibody that binds to the same epitopeas 56E11, which is the antibody that contains a heavy chain set forth inSEQ ID NO:452 and a light chain set forth in SEQ ID NO:453. In anotherexample, provided herein is an antibody that binds to the same epitopeas 17C9, which is the antibody that contains a heavy chain set forth inSEQ ID NO:454 and a light chain set forth in SEQ ID NO:455. In anotherexample, provided herein is an antibody that binds to the same epitopeas 69F6, which is the antibody that contains a heavy chain set forth inSEQ ID NO:456 and a light chain set forth in SEQ ID NO:457. Typically,such antibodies contain a variable heavy (V_(H)) chain and a variablelight (V_(L)) chain or antigen-binding fragments thereof.

The antibodies or antigen binding fragments provided herein exhibit abinding affinity constant (K_(a)) for the RSV F protein epitope of atleast or about 1×10⁸ M⁻¹, at least or about 2.5×10⁸ M⁻¹, at least orabout 5×10⁸ M⁻¹, at least or about 1×10⁹ M⁻¹, at least or about 5×10⁹M⁻¹, at least or about 1×10¹⁰ M⁻¹, at least or about 5×10¹⁰ M⁻¹, atleast or about 1×10¹¹ M⁻¹, at least or about 5×10¹¹ M⁻¹, at least orabout 1×10¹² M⁻¹, at least or about 5×10¹² M⁻¹, at least or about 1×10¹³M⁻¹, at least or about 5×10¹³ M⁻¹, at least or about 1×10¹⁴ M⁻¹, atleast or about 5×10¹⁴M⁻¹, at least or about 1×10¹⁵M⁻¹, or at least orabout 5×10¹⁵M⁻¹. The antibodies provided herein can exhibit a bindingaffinity for a recombinantly purified F protein, such as theextracellular domain of RSV A2 strain F protein set forth in SEQ IDNO:25. The antibodies provided herein also can exhibit a bindingaffinity for native RSV F protein, such as is generated by infection andexpression of RSV in cells. The antibodies provided herein can havebinding affinities that are the same or different for recombinant Fprotein versus native RSV F protein. For example, Example 5 shows that30D8 has a higher binding affinity for recombinant F protein than fornative RSV F protein. In contrast, sc5 (see e.g. U.S. Patent publicationNo. US2011/0076268 and International Published PCT Application No.WO2011/020079) exhibits similar binding affinity whether the RSV Fprotein is native or is recombinant F protein.

In some examples, the antibodies or antigen binding fragments providedherein have a dissociation constant (K_(d)) for the RSV F proteinepitope of less than or about 1×10⁻⁸ M, less than or about 4×10⁻⁹ M,less than or about 2×10⁻⁹ M, less than or about 1×10⁻⁹ M, less than orabout 2×10⁻¹⁰ M, less than or about 1×10⁻¹⁰ M, less than or about2×10⁻¹¹ M, less than or about 1×10⁻¹¹ M, less than or about 2×10⁻¹² M,less than or about 1×10⁻¹² M, less than or about 2×10⁻¹³ M, less than orabout 1×10¹³ M, less than or about 2×10⁻¹⁴ M, less than or about 1×10⁻¹⁴M, less than or about 2×10⁻¹⁵ M, less than or about 1×10⁻¹⁸ M, or lessthan or about 2×10⁻¹⁶ M.

In some examples, the antibodies or antigen-binding fragments providedherein have an EC₅₀ of less than or about 0.005 nM, less than or about0.01 nM, less than or about 0.025 nM, less than or about 0.05 nM, lessthan or about 0.075 nM, less than or about 0.1 nM, less than or about0.5 nM, less than or about 0.75 nM, less than or about 1 nM, less thanor about less than or about 1.25 nM, less than or about 1.5 nM, lessthan or about 1.75 nM, or less than or about 2 nM in an in vitromicroneutralization assay for neutralization of RSV. In particularexamples, the isolated anti-RSV antibodies or antigen-binding fragmentsprovided herein have an EC₅₀ for neutralization of RSV in an in vitroplaque reduction assay of less than or about 0.005 nM to less than orabout 2 nM; less than or about 0.005 nM to less than or about 1 nM; lessthan or about 0.005 nM to less than or about 0.5 nM; less than or about0.01 nM to less than or about 1 nM; less than or about 0.05 nM to lessthan or about 1 nM; less than or about 0.05 nM to less than or about 0.5nM; or less than or about 0.1 nM to less than or about 0.5 nM.

In some examples, an anti-RSV antibody or antigen-binding fragmentthereof provided herein neutralizes monoclonal antibody escape mutants(MARMs) against various anti-RSV antibodies in an in vitromicroneutralization assay for neutralization of RSV. In a particularexample, an anti-RSV antibody or antigen-binding fragment thereofprovided herein neutralizes a MARM with an EC₅₀ for neutralization ofthat is or is about the same as the EC₅₀ for neutralization of aparental RSV strain from which the MARM was generated. If a firstantibody can neutralize a MARM generated against a second antibody, onecan conclude that the antibodies specifically bind to or interact withdifferent epitopes.

In some examples, an anti-RSV antibody or antigen-binding fragmentthereof provided herein binds to an epitope that is less susceptible tovariation in the form of generated escape mutants (MAR MS) In someexamples, RSV is incapable of generating an escape mutant after 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more successive rounds ofviral replication in the presence of an anti-RSV antibody orantigen-binding fragment thereof provided herein. In a particularexample, RSV does not generate an escape mutant after 11, 12, 13, 14, 15or more successive rounds of viral replication in the presence of ananti-RSV antibody or antigen-binding fragment thereof provided herein.In another particular example, RSV is unable to effect escape from ananti-RSV antibody or antigen-binding fragment thereof provided herein.For example, Example 11A shows that after 12 rounds of viral replicationin the presence of 30D8, RSV was unable to effect escape. In contrast,RSV was able to escape MOTAVIZUMAB® after only 7 rounds of viralreplication.

In some examples, an anti-RSV antibody or antigen-binding fragmentthereof provided herein inhibits the binding of RSV to its host cellreceptor by at least or about 99%, at least or about 95%, at least orabout 90%, at least or about 85%, at least or about 80%, at least orabout 75%, at least or about 70%, at least or about 65%, at least orabout 60%, at least or about 55%, at least or about 50%, at least orabout 45%, at least or about 40%, at least or about 35%, at least orabout 30%, at least or about 25%, at least or about 20%, at least orabout 15%, or at least or about 10% relative to the binding of RSV toits host cell receptor in the absence of the anti-RSV antibody orantigen-binding fragment thereof. In some examples, an anti-RSV antibodyor antigen-binding fragment provided herein inhibits RSV replication byat least or about 99%, at least or about 95%, at least or about 90%, atleast or about 85%, at least or about 80%, at least or about 75%, atleast or about 70%, at least or about 65%, at least or about 60%, atleast or about 55%, at least or about 50%, at least or about 45%, atleast or about 40%, at least or about 35%, at least or about 30%, atleast or about 25%, at least or about 20%, at least or about 15%, or atleast or about 10% relative to RSV replication in the absence of theanti-RSV antibody or antigen-binding fragment thereof.

In some examples the antibodies or antigen-binding fragments thereofprovided herein have a half-life of 15 days or longer, 20 days orlonger, 25 days or longer, 30 days or longer, 40 days or longer, 45 daysor longer, 50 days or longer, 55 days or longer, 60 days or longer, 3months or longer, 4 months or longer or 5 months or longer. Methods toincrease the half-life of an antibody or antigen-binding fragmentthereof provided herein are known in the art. Such methods include forexample, pegylation, glycosylation, and amino acid substitution asdescribed elsewhere here herein.

a. Derivative Antibodies

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be used to generate derivative antibodies such as a chimericantibodies or other antigen-binding fragments, such as for example, Fab,Fab′, F(ab′)₂, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd′fragments. Generally, the derivative antibody or antigen-bindingfragment derived from a parent antibody retains the binding specificityof the parent antibody. Antibody fragments can be generated by anytechniques known to those of skill in the art. For example, Fab andF(ab′)₂ fragments can be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments), F(ab′)₂ fragmentscontain the variable region, the light chain constant region and theC_(H)1 domain of the heavy chain. Further, anti-RSV antibodies orantigen-binding fragments thereof provided herein also can be generatedusing various phage display methods known in the art. In some examples,the antigen-binding variable regions of the anti-RSV antibodies orantigen-binding fragments thereof provided herein can be recombinantlyfused to one or more constant regions known in the art to generatechimeric full length antibodies, Fab, Fab′, F(ab′)₂ or otherantigen-binding fragments. Exemplary methods for generating full lengthantibodies from antibody fragments are known in the art and providedherein. Methods for producing chimeric antibodies are known in the art(see e.g., Morrison (1985) Science 229:1202; Oi et al. (1986)BioTechniques 4:214; Gillies et al. (1989) J. Immunol. Methods125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397).

Chimeric antibodies comprising one or more CDRs from an anti-RSVantibody provided herein and framework regions from a heterologousimmunoglobulin molecule can be produced using a variety of techniquesknown in the art including, for example, CDR-grafting (EP 239,400; PCTpublication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101,and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan(1991) Molecular Immunology 28(4/5):489-498; Studnicka et al. (1994)Protein Engineering 7(6):805-814; and Roguska et al. (1994) PNAS91:969-973), and chain shuffling (U.S. Pat. No. 5,565,332).

In some examples, antibodies contain one or more CDRs of 30D8 (e.g., oneor more CDRs set forth in SEQ ID NOS:405-410 and 437) and a heterologousframework region. In some examples, antibodies contain one or more CDRsof 104E5 (e.g., one or more CDRs set forth in SEQ ID NOS:411-416 and438) and a heterologous framework region. In some examples, antibodiescontain one or more CDRs of 38F10 (e.g., one or more CDRs set forth inSEQ ID NOS:417-422 and 439) and a heterologous framework region. In someexamples, antibodies contain one or more CDRs of 14G3 (e.g., one or moreCDRs set forth in SEQ ID NOS:423-428 and 440) and a heterologousframework region. In some examples, antibodies contain one or more CDRsof 90D3 (e.g., one or more CDRs set forth in SEQ ID NOS:429-434 and 441)and a heterologous framework region. In some examples, antibodiescontain one or more CDRs of 56E11 (e.g., one or more CDRs set forth inSEQ ID NOS:458-463 and 482) and a heterologous framework region. In someexamples, antibodies contain one or more CDRs of 17C9 (e.g., one or moreCDRs set forth in SEQ ID NOS:464-469 and 483) and a heterologousframework region. In some examples, antibodies contain one or more CDRsof 69F6 (e.g., one or more CDRs set forth in SEQ ID NOS:470-475 and 484)and a heterologous framework region. Framework residues in the frameworkregions can be substituted with the corresponding residue from the CDRdonor antibody to alter, such as improve, antigen-binding. Theseframework substitutions are identified by methods well known in the art,e.g., by modeling of the interactions of the CDR and framework residuesto identify framework is residues important for antigen-binding andsequence comparison to identify unusual framework residues at particularpositions (see, e.g., U.S. Pat. No. 5,585,089; and Riechmann et al.(1988) Nature 332:323).

In some examples, the derivative anti-RSV antibodies or antigen-bindingfragments thereof have a binding affinity constant (K_(a)) for the RSV Fprotein epitope of at least or about 1×10⁸ M⁻¹, at least or about2.5×10⁸ M⁻¹, at least or about 5×10⁸ M⁻¹, at least or about 1×10⁹ M⁻¹,at least or about 5×10⁹ M⁻¹, at least or about 1×10¹⁰ M⁻¹, at least orabout 5×10¹⁰ M⁻¹, at least or about 1×10¹¹ M⁻¹, at least or about 5×10¹¹M⁻¹, at least or about 1×10¹² M⁻¹, at least or about 5×10¹² M⁻¹, atleast or about 1×10¹³ M⁻¹, at least or about 5×10¹³ M⁻¹, at least orabout 1×10¹⁴ M⁻¹, at least or about 5×10¹⁴ M⁻¹, at least or about 1×10¹⁵M⁻¹, or at least or about 5×10¹⁵ M⁻¹.

In some examples, the derivative anti-RSV antibodies or antigen-bindingfragments thereof have a dissociation constant (K_(d)) for the RSV Fprotein epitope of less than or about 1×10⁻⁸ M, less than or about4×10⁻⁹ M, less than or about 2×10⁻⁹ M, less than or about 1×10⁻⁹ M, lessthan or about 2×10⁻¹⁰ M, less than or about 1×10⁻¹⁰ M, less than orabout 2×10⁻¹¹ M, less than or about 1×10⁻¹¹ M, less than or about2×10⁻¹² M, less than or about 1×10⁻¹² M, less than or about 2×10⁻¹³ M,less than or about 1×10⁻¹³ M, less than or about 2×10⁻¹⁴ M, less than orabout 1×10⁻¹⁴ M, less than or about 2×10⁻¹⁵ M, less than or about1×10⁻¹⁵ M, or less than or about 2×10⁻¹⁶ M.

In some examples, the derivative anti-RSV antibodies or antigen-bindingfragments thereof neutralize monoclonal antibody escape mutants (MARMs)against various anti-RSV antibodies in an in vitro microneutralizationassay for neutralization of RSV. In a particular example, an anti-RSVantibody or antigen-binding fragment thereof provided herein neutralizesa MARM with an EC₅₀ for neutralization of that is or is about the sameas the EC₅₀ for neutralization of a parental RSV strain from which theMARM was generated.

In some examples, the derivative anti-RSV antibodies or antigen-bindingfragments thereof bind to an epitope that is less susceptible tovariation in the form of generated escape mutants. In some examples, RSVis incapable of generating an escape mutant after 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more successive rounds of viralreplication in the presence of a derivative anti-RSV antibody orantigen-binding fragment thereof provided herein. In another particularexample, RSV is unable to effect escape from a derivative anti-RSVantibody or antigen-binding fragment thereof provided herein.

In some examples, the derivative anti-RSV antibodies or antigen-bindingfragments thereof have EC₅₀ of less than or about 0.005 nM, less than orabout 0.01 nM, less than or about 0.025 nM, less than or about 0.05 nM,less than or about 0.075 nM, less than or about 0.1 nM, less than orabout 0.5 nM, less than or about 0.75 nM, less than or about 1 nM, lessthan or about 1.25 nM, less than or about 1.5 nM, less than or about1.75 nM, or less than or about 2 nM in an in vitro microneutralizationassay for neutralization of RSV. In particular examples, the derivativeanti-RSV antibodies or antigen-binding fragments thereof have an EC₅₀for neutralization of RSV in an in vitro plaque reduction assay of lessthan or about 0.005 nM to less than or about 2 nM; less than or about0.005 nM to less than or about 1 nM; less than or about 0.005 nM to lessthan or about 0.5 nM; less than or about 0.01 nM to less than or about 1nM; less than or about 0.05 nM to less than or about 1 nM; less than orabout 0.05 nM to less than or about 0.5 nM; or less than or about 0.1 nMto less than or about 0.5 nM.

Any derivative of an anti-RSV antibody or antigen-binding fragmentthereof provided herein can be used in therapeutic regimens, prophylaxistherapies and/or diagnostic techniques, such as in the methods provided.For example, the derivative antibodies or antigen-binding fragmentsthereof can be used to bind to RSV for the treatment, prevention and/ordetection of RSV infection or alleviation of one or more symptoms of aRSV infection.

i. Single Chain Antibodies

In particular examples, the anti-RSV antibody is a single chainantibody. A single-chain antibody can be generated from theantigen-binding domains of any of the anti-RSV antibodies orantigen-binding fragments thereof provided herein. Methods forgenerating single chain antibodies using recombinant techniques areknown in the art, such as those described in, for example, Marasco etal. (1993) Proc. Natl. Acad. Sci. USA 90:7889-7893, Whitlow and Filpula(1991) Methods, 2: 97-105; Bird et al. (1988) Science 242:423-426; Packet al. (1993) Bio/Technology 11:1271-77; and U.S. Pat. Nos. 4,946,778,5,840,300, 5,667,988, 5,658,727.

A single chain antibody can contain a light chain variable (V_(L))domain or functional region thereof and a heavy chain variable (V_(H))domain or functional region thereof of any anti-RSV antibody orantigen-binding fragment thereof provided herein. In some examples, theV_(L) domain or functional region thereof of the single chain antibodycontains a complementarity determining region 1 (CDR1), acomplementarity determining region 2 (CDR2) and/or a complementaritydetermining region 3 (CDR3) of an anti-RSV antibody or antigen-bindingfragment thereof provided herein. In some examples, the V_(H) domain orfunctional region thereof of the single chain antibody contains acomplementarity determining region 1 (CDR1), a complementaritydetermining region 2 (CDR2) and a complementarity determining region 3(CDR3) of any anti-RSV antibody or antigen-binding fragment thereofprovided herein. In some examples, the single chain antibody furthercontains a peptide linker. In such examples, a peptide linker can belocated between the light chain variable domain (V_(L)) and the heavychain variable domain (V_(H)).

The single chain antibody can contain a peptide spacer, or linker,between the one or more domains of the antibody. For example, the lightchain variable domain (V_(L)) of an antibody can be coupled to a heavychain variable domain (V_(H)) via a flexible linker peptide. Variouspeptide linkers are well-known in the art and can be employed in theprovided methods. A peptide linker can include a series of glycineresidues (Gly) or Serine (Ser) residues. Exemplary of polypeptidelinkers are peptides having the amino acid sequences (Gly-Ser)_(n),(Gly_(m)Ser)_(n) or (Ser_(m)Gly)_(n), in which m is 1 to 6, generally 1to 4, and typically 2 to 4, and n is 1 to 30, or 1 to 10, and typically1 to 4, with some glutamic acid (Glu) or lysine (Lys) residues dispersedthroughout to increase solubility (see, e.g., International PCTapplication No. WO 96/06641, which provides exemplary linkers for use inconjugates). Exemplary peptide linkers include, but are not limited topeptides having the sequence GGSSRSSSSGGGGSGGGG (SEQ ID NO:267),GSGRSGGGGSGGGGS (SEQ ID NO:268), EGKSSGSGSESKST (SEQ ID NO:269),EGKSSGSGSESKSTQ (SEQ ID NO:270), EGKSSGSGSESKVD (SEQ ID NO:271).GSTSGSGKSSEGKG (SEQ ID NO:272). KESGSVSSEQLAQFRSLD (SEQ ID NO:273), andESGSVSSEELAFRSLD (SEQ ID NO:274). Generally, the linker peptides areapproximately 1-50 amino acids in length. The linkers used herein alsocan increase intracellular availability, serum stability, specificityand solubility or provide increased flexibility or relieve sterichindrance. Linking moieties are described, for example, in Huston et al.(1988) Proc Natl Acad Sci USA 85:5879-5883. Whitlow et al. (1993)Protein Engineering 6:989-995, and Newton et al., (1996) Biochemistry35:545-553. Other suitable peptide linkers include any of thosedescribed in U.S. Pat. No. 4,751,180 or 4,935,233, which are herebyincorporated by reference.

ii. Anti-Idiotypic Antibodies

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be utilized to generate anti-idiotype antibodies that “mimic”the RSV F protein antigen, to which the antibody immunospecificallybinds, using techniques well known to those skilled in the art (see,e.g., Greenspan & Bona (1989) FASEB J. 7(5):437-444; and Nissinoff(1991) J. Immunol. 147(8):2429-2438). For example, the anti-RSVantibodies or antigen-binding fragments thereof provided herein whichbind to and competitively inhibit the binding of RSV to its host cellreceptor, as determined by assays well known in the art, can be used togenerate anti-idiotypes that “mimic” a RSV antigen and bind to the RSVreceptors, i.e., compete with the virus for binding to the host cell,therefore decreasing the infection rate of host cells with virus. Insome examples, anti-anti-idiotypes can be generated by techniqueswell-known to the skilled artisan. The anti-anti-idiotypes mimic thebinding domain of the anti-RSV antibody or antigen-binding fragmentthereof and, as a consequence, bind to and neutralize RSV.

iii. Multi-Specific Antibodies and Antibody Multimerization

Two or more antibodies or antigen-binding fragments thereof providedherein can be engineered to form multivalent derivative antibodies, ormultimers, such as bivalent, trivalent, tetravalent, pentavalent,hexavalent, heptavalent, or greater valency (i.e., containing 2, 3, 4,5, 6, 7 or more antigen-binding sites) derivative antibodies. Suchmultivalent derivative antibodies can be monospecific, bispecific,trispecific or of greater multispecificity. In some examples, themultivalent derivative antibodies are monospecific, containing two ormore antigen-binding domains that immunospecifically bind to the sameepitope. In some examples, the multivalent derivative antibodies aremultispecific, containing two or more antigen-binding domains thatimmunospecifically bind to two or more different epitopes. In someparticular examples, the multivalent derivative antibodies are bivalent,containing two antigen-binding domains. Such bivalent antibodies can behomobivalent or heterobivalent antibodies, which immunospecifically bindto the same or different epitopes, respectively.

In some examples, the multispecific antibodies can immunospecificallybind to two or more different epitopes of RSV. Techniques forengineering multispecific antibodies are known in the art, and include,for example, linkage of two or more antigen-binding fragments viacovalent, non-covalent, or chemical linkage. In some instances,multivalent derivative antibodies can be formed by dimerization of twoor more anti-RSV antibodies or antigen-binding fragments thereof.Multimerization between two anti-RSV antibodies or antigen-bindingfragments can be spontaneous, or can occur due to forced linkage of twoor more polypeptides. In one example, multimers of anti-RSV antibodiescan be linked by disulfide bonds formed between cysteine residues ondifferent anti-RSV antibodies. In another example, multivalentderivative antibodies can include anti-RSV antibodies or antigen-bindingfragments thereof joined via covalent or non-covalent interactions topeptide moieties fused to the antibody or antigen-binding fragmentthereof. Such peptides can be peptide linkers (spacers), or peptidesthat have the property of promoting multimerization. In some examples,multivalent derivative antibodies can be formed between two antibodiesthrough chemical linkage, such as for example, by usingheterobifunctional linkers.

Any multispecific and/or multivalent derivative antibody can begenerated from the anti-RSV antibodies or antigen-binding fragmentsthereof provided herein provided that the antibody is biocompatible(e.g., for administration to animals, including humans) and maintainsits activity, such as the binding to one or more epitopes of and/orneutralization of RSV. For the multispecific and multivalent derivativeantibodies provided herein, the derivative antibody is at leastimmunospecific for an epitope recognized by 30D8, 104E5, 38F10, 14G3,90D3, 56E11, 17C9 or 69F6. The multispecific and multivalent derivativeantibody provided herein also can be immunospecific for the epitoperecognized by 58c5 or sc5.

In some examples, the multispecific and/or multivalent antibody containsa V_(H) CDR1 having the amino acid sequence set forth in SEQ ID NOS:2,10, 405, 411, 417, 423, 429, 435-441, 458, 464, 470 or 482-484, a V_(H)CDR2 having the amino acid sequence set forth in SEQ ID NOS:3, 11, 406,412, 418, 424, 430, 459, 465 or 471, a V_(H) CDR3 having the amino acidsequence set forth in SEQ ID NOS:4, 12, 407, 413, 419, 425, 431, 460,466 or 472, a V_(L) CDR1 having the amino acid sequence set forth in SEQID NOS:6, 14, 408, 414, 420, 426, 432, 461, 467 or 473, a V_(L) CDR2having the amino acid sequence set forth in SEQ ID NOS:7, 15, 409, 415,421, 427, 433, 462, 468 or 474, a V_(L) CDR3 having the amino acidsequence set forth in SEQ ID NOS:8, 16, 410, 416, 422, 428, 434, 463,469 or 475, or any combination thereof.

In some examples, multispecific antibodies can be generated thatimmunospecifically bind to two or more epitopes of a RSV F protein(e.g., a RSV F protein having an amino acid sequence set forth in SEQ IDNO:282, 382 or 485). For example, the multispecific antibodies canimmunospecifically bind to two or more different epitopes in the A, B orC antigenic regions of a RSV F protein. In some examples, multispecificantibodies can be generated that immunospecifically bind to an epitopeof a RSV F protein and another RSV epitope. For example, themultispecific antibodies can immunospecifically bind to an epitope of aRSV F protein and an epitope of another RSV surface glycoprotein. Insome examples, the multispecific antibodies can immunospecifically bindto an epitope of a RSV F protein and an epitope of a RSV proteinselected from among a RSV attachment protein (e.g. having an amino acidsequence set forth in SEQ ID NO:275), a RSV RNA polymerase beta subunitlarge structural protein (L protein) (e.g. having an amino acid sequenceset forth in SEQ ID NO:276), a RSV nucleocapsid protein (e.g. having anamino acid sequence set forth in SEQ ID NO:277), a RSV nucleoprotein (N)(e.g. having an amino acid sequence set forth in SEQ ID NO:278), a RSVphosphoprotein P (e.g. having an amino acid sequence set forth in SEQ IDNO:279), a RSV matrix protein (e.g. having an amino acid sequence setforth in SEQ ID NO:280), a RSV small hydrophobic (SH) protein (e.g.having an amino acid sequence set forth in SEQ ID NO:281), a RSVRNA-dependent polymerase, a RSV G protein (e.g. having an amino acidsequence set forth in SEQ ID NO:282), or an allelic variant of any ofthe above. In some examples, the multispecific antibodies canimmunospecifically bind to an epitope of a RSV F protein and an epitopeof a RSV G protein.

In some examples, the multispecific antibody contains an anti-RSVantigen-binding fragment derived from 30D8, 104E5, 38F10, 14G3, 90D3,56E11, 17C9 or 69F6 and an anti-RSV antigen-binding fragment derivedfrom another anti-RSV antibody. For example, the other anti-RSV antibodycan be an antibody or antigen-binding fragment derived from 58c5 or sc5.In some examples, the multispecific antibody contains an anti-RSVantigen-binding fragment derived from 30D8, 104E5, 38F10, 14G3, 90D3,56E11, 17C9 or 69F6 and an anti-RSV antigen-binding fragment derivedfrom an anti-RSV antibody selected among palivizumab (SYNAGIS®), andderivatives thereof such as, but not limited to, motavizumab (NUMAX®),AFFF, P12f2, P12f4, P11d4, Ale % A12a6, A13c4, A17d4, A4B4, A8c7,1X-493L1, FR H3-3F4, M3H9, Y10H6, DG, AFFF(1), 6H8, L1-7E5, L2-15B10,A13a11, A1h5, A4B4(1), A4B4L1FR-S28R, A4B4-F52S (see, e.g., U.S. Pat.Nos. 5,824,307 and 6,818,216). In some examples, the multispecificantibody contains an anti-RSV antigen-binding fragment derived from30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 or 69F6 and an anti-RSVantigen-binding fragment derived from a human anti-RSV antibody, suchas, but not limited to, rsv6, rsv11, rsv13, rsv19 (i.e. Fab 19), rsv21,rsv22, rsv23. RF-1, and RF-2 (see, e.g. U.S. Pat. Nos. 6,685,942 and5,811,524). In some examples, the multispecific antibody contains ananti-RSV antigen-binding fragment derived from 30D8, 104E5, 38F10, 14G3,90D3, 56E11, 17C9 or 69F6 and an anti-RSV antigen-binding fragmentderived from an anti-RSV mouse monoclonal antibody such as, but notlimited to, MAbs 1153, 1142, 1200, 1214, 1237, 1129, 1121, 1107, 1112,1269, 1269, 1243 (Beeler et al. (1989) J. Virology 63(7):2841-2950).MAb151 (Mufson et al. (1987) J. Clin. Microbiol. 25:1635-1539). MAbs43-1 and 13-1 (Fernie et al. (1982) Proc. Soc. Exp. Biol. Meal.171:266-271). MAbs 1436C, 1302A, 1308F, and 13311-1 (Anderson el al(1984) J. Clin. Microbiol. 19:934-936), and humanized derivativesthereof. Additional exemplary antibodies or antigen-binding fragmentsthereof that can be used to generate a multispecific antibody thatcontains an anti-RSV antigen-binding fragment derived from 30D8, 104E5,38F10, 14G3, 90D3, 56E11, 17C9 or 69F6 include, but are not limited to,anti-RSV antibodies or antigen-binding fragments thereof described in,for example, U.S. Pat. Nos. 6,413,771, 5,840,298, 5,811,524, 6,656,467,6,537,809, 7,364,742, 7,070,786, 5,955,364, 7,488,477, 6,818,216,5,824,307, 7,364,737, 6,685,942, and 5,762,905 and U.S. Patent Pub. Nos.2007-0082002, 2005-0175986, 2004-0234528, 2006-0198840, 2009-0110684,2006-0159695, 2006-0013824, 2005-0288491, 2005-0019758, 2008-0226630,2009-0137003, and 2009-0092609. Tables 2A and 2B below set forth SEQ IDNOS for anti-RSV antibody heavy chains (Table 2A) and light chains(Table 2B). Included are full length sequences of heavy and lightchains, sequences for heavy and light variable domains, and sequences ofCDRH1, CDRH2, CDRH3, CDRL1, DRL2 and/or CDRL3, where indicated.

TABLE 2A Anti-RSV Antibody Heavy Chain SEQ ID NOS Heavy Variable ChainDomain CDR1 CDR2 CDR3 Synagis 103 104 105 106 107 AFFF 113 114 115 116P1212 122 123 124 125 126 P12f4 131 132 133 P11d4 137 138 139 Ale9 144145 A12a6 149 150 151 A13c4 155 156 157 A17d4 161 162 163 A4B4 167 168A8c7 172 173 IX-493L1FR 176 232 M3H9 179 Y10H6 185 DG 186 187 188 AFFF(1) 190 6H8 194 L1-7E5 198 L2-15B10 201 A13a11 205 206 A1h5 210 A4B4 (1)215 A4B4L1 219 A4B4-F25S 222 H3-3F4 226 227 A3e2 233 234 235 A14a4 239240 241 A16b4 246 247 A17b5 252 253 A17f5 257 258 259 Rsv6/11/21/22 362363 364 365 Rsv13/19H 366 367 368 369 Rsv23 362 363 364 365

TABLE 2B Anti-RSV Antibody Light Chain SEQ ID NOS Light Variable ChainDomain CDR1 CDR2 CDR3 Synagis 108 109 110 111 112 AFFF 117 118 119 120121 P1212 127 128 129 130 P12f4 134 135 136 P11d4 140 141 142 143 Ale9146 147 148 A12a6 152 153 154 A13c4 158 159 160 A17d4 164 165 166 A4B4169 170 171 A8c7 174 175 IX-493L1FR 177 178 M3H9 180 181 231 Y10H6 183184 185 DG 189 AFFF (1) 191 192 193 6H8 195 196 197 L1-7E5 199 200L2-15B10 202 203 204 A13a11 207 208 209 A1h5 211 212 213 214 A4B4 (1)216 217 218 A4B4L1 220 221 A4B4-F25S 223 224 225 H3-3F4 228 229 A3e2 236237 238 A14a4 242 243 244 245 A16b4 248 249 250 251 A17b5 254 255 256A17f5 260 261 262 A17h4 263 264 265 266 Rsv6/11/21/22 370 371 372 373Rsv13/19H 378 379 380 381 Rsv23 374 375 376 377

In some examples, multispecitic antibodies or antigen-binding fragmentscan immunospecitically bind to an epitope of a RSV F protein and anepitope of another heterologous polypeptide or other antigenic material,such as, for example, a solid support material (see, e.g., InternationalPCT Pub. Nos. WO 93/17715, WO 92/08802. WO 91/00360, and WO 92/05793;U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and5,601,819; Tutt, et al., (1991) J. Immunol. 147:60-69; and Kostelny etal., (1992) J. Immunol. 148:1547-1553.

(1) Multimerization Via Peptide Linkers

Peptide linkers can be used to produce multivalent antibodies, such as,for example, a multimer where one multimerization partner is an anti-RSVantibody or antigen-binding fragment thereof provided herein. In oneexample, peptide linkers can be fused to the C-terminal end of a firstpolypeptide and the N-terminal end of a second polypeptide. Thisstructure can be repeated multiples times such that at least one, suchas 2, 3, 4, or more soluble polypeptides are linked to one another viapeptide linkers at their respective termini. For example, a multimerpolypeptide can have a sequence Z₁-X-Z₂, where Z₁ and Z₂ are each asequence of an anti-RSV antigen-binding fragment (e.g. an anti-RSVsingle chain antibody; see, e.g., U.S. Pat. No. 6,759,518, describingmultimerization of single chain antibodies) and where X is a sequence ofa peptide linker. In some instances, Z₁ and/or Z₂ is an anti-RSVantigen-binding fragment provided herein. In another example, Z₁ and Z₂are different anti-RSV antigen-binding fragments, where at least Z₁ orZ₂ is derived from anti-RSV antibody or antigen-binding fragmentprovided herein. In some examples, the multimer polypeptide has asequence of Z₁-X-Z₂-(X-Z)_(n), where “n” is any integer, i.e. generally1 or 2. Typically, the peptide linker is of sufficient length to alloweach anti-RSV antigen-binding fragment to bind its respective epitopewithout interfering with binding specificity of the antibody.

(2) Multimerization Via Heterobifunctional Linking Agents

Linkage of an anti-RSV antibody or antigen-binding fragment thereofprovided herein to another anti-RSV antibody or antigen-binding fragmentto create a multivalent antibody can be direct or indirect. For example,linkage of two or more anti-RSV antibodies or antigen-binding fragmentscan be achieved by chemical linkage or facilitated by heterobifunctionallinkers, such as any known in the art or provided herein.

Numerous heterobifunctional cross-linking reagents that are used to formcovalent bonds between amino groups and thiol groups and to introducethiol groups into proteins are known to those of skill in this art (see,e.g., the PIERCE CATALOG, ImmunoTechnology Catalog & Handbook,1992-1993, which describes the preparation of and use of such reagentsand provides a commercial source for such reagents; see, also, e.g.,Cumber et al., (1992) Bioconjugate Chem. 3:397-401; Thorpe et al.,(1987) Cancer Res. 47:5924-5931; Gordon et al., (1987) Proc. Natl. AcadSci. 84:308-312; Walden et al., (1986) J. Mol. Cell Immunol. 2:191-197;Carlsson et al., (1978) Biochem. J. 173: 723-737; Mahan et al., (1987)Anal. Biochem. 162:163-170; Wawryznaczak et al., (1992) Br. J. Cancer66:361-366; Fattom et al, (1992) Infection & Immun. 60:584-589). Thesereagents can be used to form covalent bonds between two antibodies orbetween each of the antibodies and a linker. Exemplary reagents include,but are not limited to: N-succinimidyl-3-(2-pyridyldithio)propionate(SPDP; disulfide linker); sulfosuccinimidyl6-[3-(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP);succinimidyloxycarbonyl-α-methyl benzyl thiosulfate (SMBT, hindereddisulfate linker); succinimidyl 6-[3-(2-pyridyldithio)propionamido]-hexanoate (LC-SPDP); sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC);succinimidyl 3-(2-pyridyldithio)butyrate (SPDB; hindered disulfide bondlinker); sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate (SAED); sulfo-succinimidyl7-azido-4-methylcoumarin-3-acetate (SAMCA);sulfosuccinimidyl-6-[alpha-methyl-alpha-(2-pyridyldithio)toluamido]-hexanoate(sulfo-LC-SMPT); 1,4-di-[3′-(2′-pyridyldithio)propion-amido]butane(DPDPB); 4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridylthio)toluene(SMPT, hindered disulfate linker);sulfosuccinimidyl-6-[α-methyl-α-(2-pyrimiyldi-thio)toluamido]hexanoate(sulfo-LC-SMPT); m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS);m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester (sulfo-MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB; thioether linker);sulfosuccinimidyl-(4-iodoacetyl)amino benzoate (sulfo-SIAB);succinimidyl-4-(p-maleimi-dophenyl)butyrate (SMPB);sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate (sulfo-SMPB); andazidobenzoyl hydrazide (ABH). In some examples, the linkers, can be usedin combination with peptide linkers, such as those that increaseflexibility or solubility or that provide for or eliminate sterichindrance. Any other linkers known to those of skill in the art forlinking a polypeptide molecule to another molecule can be employed.

(3) Polypeptide Multimerization Domains

Interaction of two or more antigen-binding fragments to form multivalentand/or multispecific derivative antibodies can be facilitated by theirlinkage, either directly or indirectly, to any moiety or otherpolypeptide that are themselves able to interact to form a stablestructure. For example, separate encoded polypeptide chains can bejoined by multimerization, whereby multimerization of the polypeptidesis mediated by a multimerization domain. Typically, the multimerizationdomain provides for the formation of a stable protein-proteininteraction between a first chimeric polypeptide and a second chimericpolypeptide. Chimeric polypeptides include, for example, linkage(directly or indirectly) of one chain (e.g., a variable heavy domainchain or variable light chain domain) of an antibody or antigen-bindingfragment thereof with a multimerization domain. Typically, themultimerization domain is linked to a heavy chain domain of the antibodyor antigen-binding fragment thereof. Such chimeric polypeptides can begenerated as a fusion proteins using recombinant techniques for fusingnucleic acid encoding the antibody chain to nucleic acid encoding themultimerization domain.

For the multivalent and/or multispecific derivative antibodies providedherein, at least one multimerization partner is an anti-RSV antibody orantigen-binding fragment thereof linked directly or indirectly to amultimerization domain. Homo- or heteromultimeric polypeptides can begenerated from co-expression of separate chimeric polypeptides. Thefirst and second chimeric polypeptides can be the same or different.

Generally, a multimerization domain includes any polypeptide capable offorming a stable protein-protein interaction with another polypeptide.The multimerization domains can interact, for example, via animmunoglobulin sequence (e.g., an Fc domain), a leucine zipper, ahydrophobic region, a hydrophilic region, or a free thiol which forms anintermolecular disulfide bond between the chimeric molecules of a homo-or heteromultimer. In addition, a multimerization domain can include anamino acid sequence comprising a protuberance complementary to an aminoacid sequence comprising a hole or pocket, such as is described, forexample, in U.S. Pat. No. 5,731,168. Such a multimerization region canbe engineered such that steric interactions not only promote stableinteraction, but further promote the formation of heterodimers overhomodimers from a mixture of chimeric monomers.

In some examples, multivalent and/or multispecific antibodies aregenerated by linkage of two anti-RSV antigen-binding fragments viamultimerization domain. In such examples, at least one of theantigen-binding fragments is derived from an anti-RSV antibody orantigen-binding fragment thereof provided herein, such as for example,58c5, sc5, 30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 or 69F6.

An antigen-binding polypeptide, such as for example anti-RSVantigen-binding fragment, can be conjugated to a multimerization domainto form a chimeric polypeptide. For anti-RSV antigen-binding fragmentscontaining more than one chain (e.g., a variable heavy domain chain anda variable light chain domain), the multimerization domain can beconjugated to one of the chains, typically the heavy chain. Theantigen-binding fragment is typically linked via its N- or C-terminus tothe N- or C-terminus of the multimerization domain. Typically, themultimerization domain is conjugated to the C-terminus of theantigen-binding fragment (e.g., the C-terminus of a single chainantibody or the C-terminus of one chain of the antigen-bindingfragment). The linkage can be direct or indirect via a linker. Also, thechimeric polypeptide can be a fusion protein or can be formed bychemical linkage, such as through covalent or non-covalent interactions.For example, when preparing a chimeric polypeptide containing amultimerization domain, nucleic acid encoding all or part of an anti-RSVantigen-binding fragment can be operably linked to nucleic acid encodingthe multimerization domain sequence, directly or indirectly oroptionally via a linker domain. Typically, the construct encodes achimeric protein where the C-terminus of the anti-RSV antigen-bindingfragment (or single chain of the antigen-binding fragment) is joined tothe N-terminus of the multimerization domain.

A multivalent antibody provided herein contains two chimeric proteinscreated by linking, directly or indirectly, two of the same or differentanti-RSV antigen-binding fragments directly or indirectly to amultimerization domain. In some examples, where the multimerizationdomain is a polypeptide, a gene fusion encoding the anti-RSVantigen-binding fragment (or single chain of the antigen-bindingfragment) multimerization domain chimeric polypeptide is inserted intoan appropriate expression vector. The resulting anti-RSV antigen-bindingfragment-multimerization domain chimeric proteins can be expressed inhost cells transformed with the recombinant expression vector, andallowed to assemble into multimers, where the multimerization domainsinteract to form multivalent antibodies. Chemical linkage ofmultimerization domains to anti-RSV antigen-binding fragments also canbe effected using heterobifunctional linkers as discussed above. In someexamples, the multivalent antibodies are multispecific antibodies thatare derived from two or more anti-RSV antigen-binding fragments whichbind to different epitopes.

The resulting chimeric polypeptides, and multivalent antibodies formedtherefrom, can be purified by any suitable method known in the art, suchas, for example, by affinity chromatography over Protein A or Protein Gcolumns. Where two nucleic acid molecules encoding different anti-RSVantigen-binding chimeric polypeptides are transformed into cells,formation of homo- and heterodimers will occur. Conditions forexpression can be adjusted so that heterodimer formation is favored overhomodimer formation.

(a) Immunoglobulin Domain

Multimerization domains include those comprising a free thiol moietycapable of reacting to form an intermolecular disulfide bond with amultimerization domain of an additional amino acid sequence. Forexample, a multimerization domain can include a portion of animmunoglobulin molecule, such as from IgG₁, IgG₂, IgG₃, IgG₄, IgA, IgD,IgM, and IgE. Generally, the portion of an immunoglobulin selected foruse as a multimerization domain is the constant region (Fe).Preparations of fusion proteins containing polypeptides fused to variousportions of antibody-derived polypeptides, including the Fe domain, havebeen described (see, e.g., Ashkenazi et al. (1991) PNAS 88: 10535; Byrnet al. (1990) Nature, 344:677; and Hollenbaugh and Aruffo, (1992)“Construction of Immunoglobulin Fusion Proteins.” in Current Protocolsin Immunology. Suppl. 4, pp. 10.19.1-10.19.11).

In humans, there are five antibody isotypes classified based on theirheavy chains denoted as delta (δ), gamma (γ), mu (μ), alpha (α) andepsilon (ε), giving rise to the IgD, IgG, IgM, IgA, and IgE classes ofantibodies, respectively. The IgA and IgG classes contain the subclassesIgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Sequence differences betweenimmunoglobulin heavy chains cause the various isotypes to differ in, forexample, the number of constant (C) domains, the presence of a hingeregion, and the number and location of interchain disulfide bonds. Forexample, IgM and IgE heavy chains contain an extra C domain (C4), thatreplaces the hinge region. The Fc regions of IgG, IgD, and IgA pair witheach other through their Cγ3, Cδ3, and Cα3 domains, whereas the Fcregions of IgM and IgE dimerize through their Cμ4 and Cε4 domains. IgMand IgA form multivalent structures with ten and four antigen-bindingsites, respectively.

Antigen-binding chimeric polypeptides provided herein includefull-length immunoglobulin polypeptides (i.e., including all domains offull-length immunoglobulins). In some examples, the antigen-bindingchimeric polypeptide is less than full length (e.g. the chimericpolypeptide can contain the antigen-binding domain and one or moreimmunoglobulin domains for multimerization, where the chimericpolypeptide is not a full-length immunoglobulin). In some examples, theanti-RSV antigen-binding chimeric polypeptides are assembled asmonovalent or hetero- or homo-multivalent antibodies, such as bivalent,trivalent, tetravalent, pentavalent, hexavalent, heptavalent or highervalency antibodies. Chains or basic units of varying structures (e.g.,one more heterologous constant regions or domains) can be utilized toassemble the monovalent and hetero- and homo-multivalent antibodies.Anti-RSV antigen-binding chimeric polypeptides can be readily producedand secreted by mammalian cells transformed with the appropriate nucleicacid molecule. In some examples, one or more than one nucleic acidfusion molecule can be transformed into host cells to produce amultivalent antibody where the anti-RSV antigen-binding portions of themultivalent antibody are the same or different. Typically, at least oneof the anti-RSV antigen-binding portions of the multivalent antibody isderived from an anti-RSV antibody or antigen-binding fragment thereofprovided herein, such as for example, 58c5, sc5, 30D8, 104E5, 38F10,14G3, 90D3, 56E11, 17C9 or 69F6.

(i) Fc Domain

Exemplary multimerization domains that can be used to generatemultivalent and/or multispecific antibodies containing an anti-RSVantigen-binding fragment provided herein include polypeptides derivedfrom a heavy chain constant region or domain of a selectedimmunoglobulin molecule. Exemplary sequences of heavy chain constantregions for human IgG sub-types are set forth in SEQ ID NOS:356 (IgG1),SEQ ID NO:357 (IgG2), SEQ ID NO:358 (IgG3), and SEQ ID NO:359 (IgG4).For example, for the exemplary heavy chain constant region set forth inSEQ ID NO:356, the C_(H)1 domain corresponds to amino acids 1-103, thehinge region corresponds to amino acids 104-119, the C_(H)2 domaincorresponds to amino acids 120-223, and the C_(H)3 domain corresponds toamino acids 224-330.

In one example, an immunoglobulin polypeptide chimeric protein caninclude the Fe region of an immunoglobulin polypeptide. Typically, sucha fusion retains at least a functionally active hinge, C_(H)2 and C_(H)3domains of the constant region of an immunoglobulin heavy chain. Forexample, a full-length Fc sequence of IgG1 includes amino acids 104-330of the sequence set forth in SEQ ID NO:356. An exemplary Fc sequence forhIgG1 is set forth in SEQ ID NO:360, and contains the hinge sequencecorresponding to amino acids 104-119 of SEQ ID NO:356, and the completesequence for the C_(H)2 and C_(H)3 domain as set forth in SEQ ID NO:356.Another exemplary Fe polypeptide is set forth in PCT application WO93/10151, and is a single chain polypeptide extending from theN-terminal hinge region to the native C-terminus of the Fc region of ahuman IgG1 antibody (SEQ ID NO:361). The precise site at which thelinkage is made is not critical: particular sites are well known in theart and can be selected in order to optimize the biological activity,secretion, or binding characteristics of the anti-RSV antigen-bindingchimeric polypeptide. For example, other exemplary Fe polypeptidesequences begin at amino acid C109 or P113 of the sequence set forth inSEQ ID NO:356 (see e.g., US 2006/0024298).

In addition to hIgG1 Fc, other Fc regions also can be included in theanti-RSV antigen-binding chimeric polypeptides provided herein. Forexample, the Fc fusions can contain immunoglobulin sequences that aresubstantially encoded by immunoglobulin genes belonging to any of theantibody classes, including, but not limited to IgG (including humansubclasses IgG1, IgG2, IgG3, or IgG4), IgA (including human subclassesIgA1 and IgA2), IgD, IgE, and IgM classes of antibodies.

In some examples, a Fc domain can be selected based on the functionalproperties of the domain, such as for example, the effector functions ofthe Fc domain in mediating an immune response. For example, whereeffector functions mediated by Fc/FcγR interactions are to be minimized,fusion with IgG isotypes that poorly recruit complement or effectorcells, such as for example, the Fe of IgG2 or IgG4, can be used.

Modified Fc domains also are contemplated herein for use in chimeraswith anti-RSV antigen-binding fragments, see e.g. U.S. Pat. No.7,217,797; and U.S. Pat. Pub. Nos. 2006/0198840, 2006/0024298 and2008/0287657; and International Patent Pub. No. WO 2005/063816 forexemplary modifications. Exemplary amino acid modification of Fc domainsalso are provided elsewhere herein.

Typically, a bivalent antibody is a dimer of two chimeric proteinscreated by linking, directly or indirectly, two of the same or differentanti-RSV antigen-binding fragments to an Fc polypeptide. In someexamples, a gene fusion encoding the chimeric protein is inserted intoan appropriate expression vector. The resulting chimeric proteins can beexpressed in host cells transformed with the recombinant expressionvector, and allowed to assemble, where interchain disulfide bonds formbetween the Fc moieties to yield divalent anti-RSV antibodies.Typically, a host cell and expression system is a mammalian expressionsystem to allow for glycosylation of the chimeric protein. The resultingchimeric polypeptides containing Fe moieties, and multivalent antibodiesformed therefrom, can be easily purified by affinity chromatography overProtein A or Protein G columns. Where two nucleic acids encodingdifferent anti-RSV chimeric polypeptides are transformed into cells, theformation of heterodimers must be biochemically achieved since anti-RSVchimeric molecules carrying the Fc-domain will be expressed asdisulfide-linked homodimers as well. Thus, homodimers can be reducedunder conditions that favor the disruption of inter-chain disulfides,but do not effect intra-chain disulfides. Typically, chimeric monomerswith different extracellular portions are mixed in equimolar amounts andoxidized to form a mixture of homo- and heterodimers. The components ofthis mixture are separated by chromatographic techniques.

Alternatively, the formation of a heterodimer can be biased bygenetically engineering and expressing anti-RSV antigen-binding fusionmolecules that contain an anti-RSV antigen-binding fragment, followed bythe Fc-domain of hIgG, followed by either c-jun or the c-fos leucinezippers. Since the leucine zippers form predominantly heterodimers, theycan be used to drive the formation of the heterodimers when desired,anti-RSV chimeric polypeptides containing Fc regions also can beengineered to include a tag with metal chelates or other epitope. Thetagged domain can be used for rapid purification by metal-chelatechromatography, and/or by antibodies, to allow for detection of westernblots, immunoprecipitation, or activity depletion/blocking in bioassays.

D. ADDITIONAL MODIFICATIONS OF ANTI-RSV ANTIBODIES

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be further modified. Modifications of an anti-RSV antibody orantigen-binding fragment can improve one or more properties of theantibody, including, but not limited to, decreasing the immunogenicityof the antibody or antigen-binding fragment, improving the half-life ofthe antibody or antigen-binding fragment, such as reducing thesusceptibility to proteolysis and/or reducing susceptibility tooxidation, and altering or improving of the binding properties of theantibody or antigen-binding fragment thereof. Exemplary modificationsinclude, but are not limited to, modifications of the primary amino acidsequence of the anti-RSV antibody or antigen-binding fragment thereofand alteration of the post-translational modification of the anti-RSVantibody or antigen-binding fragment thereof. Exemplarypost-translational modifications include, for example, glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization withprotecting/blocking group, proteolytic cleavage, linkage to a cellularligand or other protein. Other exemplary modifications includeattachment of one or more heterologous peptides to the anti-RSV antibodyor antigen-binding fragment to alter or improve one or more propertiesof the antibody or antigen-binding fragment thereof.

Generally, the modifications do not result in increased immunogenicityof the antibody or antigen-binding fragment thereof or significantlynegatively affect the binding of the antibody or antigen-bindingfragment thereof to RSV. Methods of assessing the binding of themodified antibodies or antigen-binding fragments thereof to a RSV Fprotein are provided herein and known in the art. For example, modifiedantibodies or antigen-binding fragments thereof can be assayed forbinding to a RSV F protein by methods such as, but not limited to,ELISA, surface plasmon resonance (SPR), or through in vitromicroneutralization assays.

Provided herein are methods of improving the half-life of any of theprovided anti-RSV antibodies or antigen-binding fragments thereof.Increasing the half-life of the anti-RSV antibodies or antigen-bindingfragments thereof provided herein can increase the therapeuticeffectiveness of the anti-RSV antibodies or antigen-binding fragmentsthereof and allow for less frequent administration of the antibodies orantigen-binding fragments thereof for prophylaxis and/or treatment, suchas preventing or treating a RSV infection, preventing, treating, and/oralleviating of one or more symptoms of a RSV infection, or reducing theduration of a RSV infection.

Modification of the anti-RSV antibodies or antigen-binding fragmentsthereof produced herein can include one or more amino acidsubstitutions, deletions or additions, either from natural mutation orhuman manipulation from the parent antibody. Methods for modification ofpolypeptides, such as antibodies, are known in the art and can beemployed for the modification of any antibody or antigen-bindingfragment thereof provided herein. In some examples, the pharmacokineticproperties of the anti-RSV antibodies or antigen-binding fragmentsthereof provided herein can be enhanced through Fc modifications bytechniques known to those skilled in the art. Standard techniques knownto those skilled in the art can be used to introduce mutations in thenucleotide molecule encoding an antibody or an antigen-binding fragmentprovided herein in order to produce an polypeptide with one or moreamino acid substitutions. Exemplary techniques for introducing mutationsinclude, but are not limited to, site-directed mutagenesis andPCR-mediated mutagenesis.

The anti-RSV antibodies and antigen-binding fragments thereof providedherein can be modified by the attachment of a heterologous peptide tofacilitate purification. Generally such peptides are expressed as afusion protein containing the antibody fused to the peptide at the C- orN-terminus of the antibody or antigen-binding fragment thereof.Exemplary peptides commonly used for purification include, but are notlimited to, hexa-histidine peptides, hemagglutinin (HA) peptides, andflag tag peptides (see e.g., Wilson et al. (1984) Cell 37:767; Witzgallet al. (1994) Anal Biochem 223:2, 291-8). The fusion does notnecessarily need to be direct, but can occur through a linker peptide.In some examples, the linker peptide contains a protease cleavage sitewhich allows for removal of the purification peptide followingpurification by cleavage with a protease that specifically recognizesthe protease cleavage site.

The anti-RSV antibodies and antigen-binding fragments thereof providedherein also can be modified by the attachment of a heterologouspolypeptide that targets the antibody or antigen-binding fragment to aparticular cell type (e.g., respiratory epithelial cells), either invitro or in vivo. In some examples an anti-RSV antibody orantigen-binding fragment thereof provided herein can be targeted to aparticular cell type by fusing or conjugating the antibody orantigen-binding fragment thereof to an antibody specific for aparticular cell surface receptor or other polypeptide that interactswith a specific cell receptor.

In some examples, an anti-RSV antibody or antigen-binding fragmentthereof provided herein can be targeted to a target cell surface and/ortaken up by the target cell by fusing or conjugating the antibody orantigen-binding fragment thereof to a peptide that binds to cell surfaceglycoproteins, such as a protein transduction domain (e.g., a TATpeptide). Exemplary protein transduction domains include, but are notlimited to, PTDs derived from proteins such as human immunodeficiencyvirus 1 (HIV-1) TAT (Ruben et al. (1989) J. Virol. 63:1-8; e.g., SEQ IDNOS:326-337, such as for example, GRKKRRQRRR (TAT 48-57) SEQ IDNO:330)), the herpes virus tegument protein VP22 (Elliott and O'Hare(1997) Cell 88:223-233; e.g., SEQ ID NO:342), the homeotic protein ofDrosophila melanogaster Antennapedia (Antp) protein (Penetratin PTD;Derossi et al. (1996) J. Biol. Chem. 271:18188-18193; e.g., SEQ IDNOS:311-314), the protegrin 1 (PG-1) anti-microbial peptide SynB (e.g.,SynB1, SynB3, and Syn B4; Kokryakov et al. (1993) FEBS Lett.327:231-236; e.g., SEQ ID NOS:323-325, respectively) and basicfibroblast growth factor (Jans (1994) FASEB J. 8:841-847; e.g., SEQ IDNO:307). PTDs also include synthetic PTDs, such as, but not limited to,polyarginine peptides (Futaki et al. (2003) J. Mol. Recognit.16:260-264; Suzuki et al. (2001) J. Biol. Chem. 276:5836-5840; e.g. SEQID NOS:315-316), transportan (Pooga et al. (1988) FASEB J. 12:67-77;Pooga et al. (2001) FASEB J. 15:1451-1453; e.g., SEQ ID NOS:338-341),MAP (Oehlke et al. (1998) Biochim. Biophys. Acta. 1414:127-139; e.g.,SEQ ID NO:305), KALA (Wyman et al. (1997) Biochemistry 36:3008-3017;e.g., SEQ ID NO:303) and other cationic peptides, such as, for example,various β-cationic peptides (Akkarawongsa et al. (2008) Antimicrob.Agents and Chemother. 52(6):2120-2129).

The anti-RSV antibodies and antigen-binding fragments thereof providedherein can be modified by the attachment of diagnostic and/ortherapeutic moiety to the antibody or antigen-binding fragment thereof.The anti-RSV antibodies and antigen-binding fragments thereof providedherein can be modified by the covalent attachment of any type ofmolecule, such as a diagnostic or therapeutic molecule, to the antibodyor antigen-binding fragment thereof such that covalent attachment doesnot prevent the antibody or antigen-binding fragment thereof frombinding to its corresponding epitope. For example, an anti-RSV antibodyor antigen-binding fragment thereof provided herein can be furthermodified by covalent attachment of a molecule such that the covalentattachment does not prevent the antibody or antigen-binding fragmentthereof from binding to RSV. In some examples, the antibodies orantigen-binding fragments thereof can be recombinantly fused to aheterologous polypeptide at the N terminus or C terminus or chemicallyconjugated, including covalent and non-covalent conjugation, to aheterologous polypeptide or other composition. For example, theheterologous polypeptide or composition can be a diagnostic polypeptideor other diagnostic moiety or a therapeutic polypeptide or othertherapeutic moiety. Exemplary diagnostic and therapeutic moietiesinclude, but are not limited to, drugs, radionucleotides, toxins,fluorescent molecules (see, e.g. International PCT Publication Nos. WO92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP396,387). Diagnostic polypeptides or diagnostic moieties can be used,for example, as labels for in vivo or in vitro detection. Therapeuticpolypeptides or therapeutic moieties can be used, for example, fortherapy of a viral infection, such as RSV infection, or for treatment ofone or more symptoms of a viral infection.

Additional fusion proteins of the anti-RSV antibodies or antigen-bindingfragments thereof provided herein can be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling can be employed to alter the activities of anti-RSV antibodiesor antigen-binding fragments thereof provided herein, for example, toproduce antibodies or antigen-binding fragments thereof with higheraffinities and lower dissociation rates (see, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal. (1997) Curr. Opinion Biotechnol. 8:724-33; Harayama (1998) TrendsBiotechnol. 16(2):76-82; Hansson et al., (1999) J. Mol. Biol.287:265-76; and Lorenzo and Blasco (1998) Biotechniques 24(2):308-13).

The provided anti-RSV antibodies or antigen-binding fragments thereofcan also be attached to solid supports, which are useful forimmunoassays or purification of the target antigen. Exemplary solidsupports include, but are not limited to, glass, cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

1. Modifications to Reduce Immunogenicity

In some examples, the antibodies or antigen-binding fragments thereofprovided herein can be further modified to reduce the immunogenicity ina subject, such as a human subject. For example, one or more amino acidsin the antibody or antigen-binding fragment thereof can be modified toalter potential epitopes for human T-cells in order to eliminate orreduce the immunogenicity of the antibody or antigen-binding fragmentthereof when exposed to the immune system of the subject. Exemplarymodifications include substitutions, deletions and insertion of one ormore amino acids, which eliminate or reduce the immunogenicity of theantibody or antigen-binding fragment thereof. Generally, suchmodifications do not alter the binding specificity of the antibody orantigen-binding fragment thereof for its respective antigen. Reducingthe immunogenicity of the antibody or antigen-binding fragment thereofcan improve one or more properties of the antibody or antigen-bindingfragment thereof, such as, for example, improving the therapeuticefficacy of the antibody or antigen-binding fragment thereof and/orincreasing the half-life of the antibody or antigen-binding fragmentthereof in vivo.

2. Fc Modifications

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can contain wild-type or modified Fc region. As describedelsewhere herein, a Fc region can be linked to an anti-RSVantigen-binding fragment provided herein, such as, for example, 58c5,sc5, 30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 or 69F6, or anantigen-binding fragment derived from 58c5, sc5, 30D8, 104E5, 38F10,14G3, 90D3, 56E11, 17C9 or 69F6. In some examples, the Fc region can bemodified to alter one or more properties of the Fe polypeptide. Forexample, the Fc region can be modified to alter (i.e. more or less)effector functions compared to the effector function of an Fc region ofa wild-type immunoglobulin heavy chain. The Fe regions of an antibodyinteract with a number of Fe receptors, and ligands, imparting an arrayof important functional capabilities referred to as effector functions.Fc effector functions include, for example, Fc receptor binding,complement fixation, and T cell depleting activity (see e.g., U.S. Pat.No. 6,136,310). Methods of assaying T cell depleting activity, Fceffector function, and antibody stability are known in the art. Forexample, the Fc region of an IgG molecule interacts with the FcγRs.These receptors are expressed in a variety of immune cells, includingfor example, monocytes, macrophages, neutrophils, dendritic cells,eosinophils, mast cells, platelets, B cells, large granular lymphocytes,Langerhans' cells, natural killer (NK) cells, and γδ T cells. Formationof the Fc/FcγR complex recruits these effector cells to sites of boundantigen, typically resulting in signaling events within the cells andimportant subsequent immune responses such as release of inflammationmediators, B cell activation, endocytosis, phagocytosis, and cytotoxicattack. The ability to mediate cytotoxic and phagocytic effectorfunctions is a potential mechanism by which antibodies destroy targetedcells. Recognition of and lysis of bound antibody on target cells bycytotoxic cells that express FcγRs is referred to as antibody dependentcell-mediated cytotoxicity (ADCC). Other Fc receptors for variousantibody isotypes include FcεRs (IgE), FcαRs (IgA), and FcγRs (IgM).

Thus, a modified Fc domain can have altered affinity, including but notlimited to, increased or low or no affinity for the Fc receptor. Forexample, the different IgG subclasses have different affinities for theFcγRs, with IgG1 and IgG3 typically binding substantially better to thereceptors than IgG2 and IgG4. In addition, different FcγRs mediatedifferent effector functions. FcγR1, FcγRIIa/c, and FcγRIIIa arepositive regulators of immune complex triggered activation,characterized by having an intracellular domain that has animmunoreceptor tyrosine-based activation motif (ITAM). FcγRIIb, however,has an immunoreceptor tyrosine-based inhibition motif (ITIM) and istherefore inhibitory. Thus, altering the affinity of an Fc region for areceptor can modulate the effector functions induced by the Fc domain.

In one example, an Fc region is used that is modified for optimizedbinding to certain FcγRs to better mediate effector functions, such asfor example, antibody-dependent cellular cytotoxicity, ADCC. Suchmodified Fe regions can contain modifications at one or more of aminoacid residues (according to the Kabat numbering scheme, Kabat et al.(1991) Sequences of Proteins of Immunological Interest, U.S. Departmentof Health and Human Services), including, but not limited to, amino acidpositions 249, 252, 259, 262, 268, 271, 273, 277, 280, 281, 285, 287,296, 300, 317, 323, 343, 345, 346, 349, 351, 352, 353, and 424. Forexample, modifications in an Fc region can be made corresponding to anyone or more of G119S, G119A, S122D, S122E, S122N, S122Q. S122T, K129H,K129Y, D132Y, R138Y, E141Y, T143H, V1471, S150E, H151D, E155Y, E155I,E155H, K157E, G164D, E166L, E166H, S181A, S181D, S187T, S207G, S3071,K209T, K209E, K209D, A210D, A213Y, A213L, A213I, 1215D, 1215E, 1215N,I215Q, E216Y, E216A, K217T, K217F, K217A, and P279L of the exemplaryIgG1 sequence set forth in SEQ ID NO:356, or combinations thereof. Amodified Fc containing these mutations can have enhanced binding to anFcR such as, for example, the activating receptor FcγIIIa and/or canhave reduced binding to the inhibitory receptor FcγRIIb (see e.g., US2006/0024298). Fc regions modified to have increased binding to FcRs canbe more effective in facilitating the destruction of viral (e.g. RSV)infected cells in patients.

In some examples, the antibodies or antigen-binding fragments providedherein can be further modified to improve the interaction of theantibody or antigen-binding fragment thereof with the FcRn receptor inorder to increase the in vivo half-life and pharmacokinetics of theantibody or antigen-binding fragment thereof (see, e.g. U.S. Pat. No.7,217,797, U.S. Pat. Pub. Nos. 2006/0198840 and 2008/0287657). FcRn isthe neonatal FcR, the binding of which recycles endocytosed antibody orantigen-binding fragment thereof from the endosomes back to thebloodstream. This process, coupled with preclusion of kidney filtrationdue to the large size of the full length molecule, results in favorableantibody serum half-lives ranging from one to three weeks. Binding of Fcto FcRn also plays a role in antibody transport.

Exemplary modifications of the Fc region include but are not limited to,mutation of the Fc described in U.S. Pat. No. 7,217,797; U.S. Pat. Pub.Nos. 2006/0198840, 2006/0024298 and 2008/0287657, and InternationalPatent Pub. No. WO 2005/063816, such as mutations at one or more ofamino acid residues (Kabat numbering. Kabat et al. (1991)) 251-256,285-90, 308-314, in the C_(H)2 domain and/or amino acids residues385-389, and 428-436 in the C_(H)3 domain of the Fc heavy chain constantregion, where the modification alters Fe receptor binding affinityand/or serum half-life relative to unmodified antibody orantigen-binding fragment thereof. In some examples, the IgG constantdomain is modified in the Fe region at one or more of amino acidpositions 250, 251, 252, 254, 255, 256, 263, 308, 309, 311, 312 and 314in the C_(H)2 domain and/or amino acid positions 385, 386, 387, 389,428, 433, 434, 436, and 459 in the C_(H)3 domain of the IgG heavy chainconstant region. Such modifications correspond to amino acids Gly120,Pro121, Ser122, Phe124, Leu125, Phe126, Thr133, Pro174, Arg175, Glu177,Gln178, and Asn180 in the C_(H)2 domain and amino acids Gln245, Va1246,Ser247, Thr249, Ser283, Gly285, Ser286, Phe288, and Met311 in the C_(H)3domain in an exemplary IgG1 sequence set forth in SEQ ID NO:356. In someexamples, the modification is at one or more surface-exposed residues,and the modification is a substitution with a residue of similar charge,polarity or hydrophobicity to the residue being substituted.

In particular examples, a Fc heavy chain constant region is modified atone or more of amino acid positions 251, 252, 254, 255, and 256 (Kabatnumbering), where position 251 is substituted with Leu or Arg, position252 is substituted with Tyr, Phe, Ser, Trp or Thr, position 254 issubstituted with Thr or Ser, position 255 is substituted with Leu, Gly,Ile or Arg, and/or position 256 is substituted with Ser, Arg, Gln, Glu,Asp, Ala, Asp or Thr. In some examples, a Fc heavy chain constant regionis modified at one or more of amino acid positions 308, 309, 311, 312,and 314, where position 308 is substituted with Thr or Ile, position 309is substituted with Pro, position 311 is substituted with serine or Glu,position 312 is substituted with Asp, and/or position 314 is substitutedwith Leu. In some examples, a Fc heavy chain constant region is modifiedat one or more of amino acid positions 428, 433, 434, and 436, whereposition 428 is substituted with Met, Thr, Leu, Phe, or Ser, position433 is substituted with Lys, Arg, Ser, Ile, Pro, Gln, or His, position434 is substituted with Phe, Tyr, or His, and/or position 436 issubstituted with His, Asn, Asp, Thr, Lys, Met, or Thr. In some examples,a Fc heavy chain constant region is modified at one or more of aminoacid positions 263 and 459, where position 263 is substituted with Glnor Glu and/or position 459 is substituted with Leu or Phe.

In some examples, a Fc heavy chain constant region can be modified toenhance binding to the complement protein C1q. In addition tointeracting with FcRs, Fc also interact with the complement protein C1qto mediate complement dependent cytotoxicity (CDC). C1q forms a complexwith the serine proteases C1r and C1s to form the C1 complex. C1q iscapable of binding six antibodies, although binding to two IgGs issufficient to activate the complement cascade. Similar to Fc interactionwith FcRs, different IgG subclasses have different affinity for C1q,with IgG1 and IgG3 typically binding substantially better than IgG2 andIgG4. Thus, a modified Fc having increased binding to C1q can mediateenhanced CDC, and can enhance destruction of viral (e.g., RSV) infectedcells. Exemplary modifications in an Fc region that increase binding toC1q include, but are not limited to, amino acid modifications atpositions 345 and 353 (Kabat numbering). Exemplary modifications includethose corresponding to K209W, K209Y, and E216S in an exemplary IgG1sequence set forth in SEQ ID NO:356.

In another example, a variety of Fc mutants with substitutions to reduceor ablate binding with FcγRs also are known. Such muteins are useful ininstances where there is a need for reduced or eliminated effectorfunction mediated by Fc. This is often the case where antagonism, butnot killing of the cells bearing a target antigen is desired. Exemplaryof such an Fc is an Fe mutein described in U.S. Pat. No. 5,457,035,which is modified at amino acid positions 248, 249 and 251 (Kabatnumbering In an exemplary IgG1 sequence set forth in SEQ ID NO:356,amino acid 117 is modified from Leu to Ala, amino acid 118 is modifiedfrom Leu to Glu, and amino acid 120 is modified from Gly to Ala. Similarmutations can be made in any Fe sequence such as, for example, theexemplary Fc sequence. This mutein exhibits reduced affinity for Fcreceptors.

The antibodies or antigen-binding fragments thereof provided herein canbe engineered to contain modified Fc regions. For example, methods forfusing or conjugating polypeptides to the constant regions of antibodies(i.e. making Fc fusion proteins) are known in the art and described in,for example, U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053,5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al.(1991) Proc. Natl. Acad. Sci. USA 88:10535-10539; Traunecker et al.(1988) Nature 331:84-86; Zheng et al. (1995) J. Immunol. 154:5590-5600;and Vil et al. (1992) Proc. Natl. Acad Sci. USA 89:11337-11341 (1992)and described elsewhere herein. In some examples, a modified Fc regionhaving one or more modifications that increases the FcRn bindingaffinity and/or improves half-life can be fused to an anti-RSV antibodyor antigen-binding fragment thereof provided herein.

3. Pegylation

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be conjugated to polymer molecules such as high molecularweight polyethylene glycol (PEG) to increase half-life and/or improvetheir pharmacokinetic profiles. Conjugation can be carried out bytechniques known to those skilled in the art. Conjugation of therapeuticantibodies with PEG has been shown to enhance pharmacodynamics while notinterfering with function (see, e.g., Deckert et al., Int. J. Cancer 87:382-390, 2000; Knight et al., Platelets 15: 409-418, 2004; Leong et al.,Cytokine 16: 106-119, 2001; and Yang et al., Protein Eng. 16: 761-770,2003). PEG can be attached to the antibodies or antigen-bindingfragments with or without a multifunctional linker either throughsite-specific conjugation of the PEG to the N- or C-terminus of theantibodies or antigen-binding fragments or via epsilon-amino groupspresent on lysine residues. Linear or branched polymer derivatizationthat results in minimal loss of biological activity can be used. Thedegree of conjugation can be monitored by SDS-PAGE and mass spectrometryto ensure proper conjugation of PEG molecules to the antibodies.Unreacted PEG can be separated from antibody-PEG conjugates by, e.g.,size exclusion or ion-exchange chromatography. PEG-derivatizedantibodies or antigen-binding fragments thereof can be tested forbinding activity to RSV antigens as well as for in vivo efficacy usingmethods known to those skilled in the art, for example, by immunoassaysdescribed herein.

4. Conjugation of a Detectable Moiety

In some examples, the anti-RSV antibodies and antibody fragmentsprovided herein can be further modified by conjugation to a detectablemoiety. The detectable moieties can be detected directly or indirectly.Depending on the detectable moiety selected, the detectable moiety canbe detected in vivo and/or in vitro. The detectable moieties can beemployed, for example, in diagnostic methods for detecting exposure toRSV or localization of RSV or binding assays for determining the bindingaffinity of the anti-RSV antibody or antigen-binding fragment thereoffor RSV. The detectable moieties also can be employed in methods ofpreparation of the anti-RSV antibodies, such as, for example,purification of the antibody or antigen-binding fragment thereof.Typically, detectable moieties are selected such that conjugation of thedetectable moiety does not interfere with the binding of the antibody orantigen-binding fragment thereof to the target epitope. Generally, thechoice of the detectable moiety depends on sensitivity required, ease ofconjugation with the compound, stability requirements, availableinstrumentation, and disposal provisions. One of skill in the art isfamiliar with labels and can identify a detectable label suitable forand compatible with the assay employed. Methods of labeling antibodieswith detectable moieties are known in the art and include, for example,recombinant and chemical methods.

The detectable moiety can be any material having a detectable physicalor chemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied in the methods provided. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labelsinclude, but are not limited to, fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), in particular, gamma and positron emittingradioisotopes (e.g., ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe), metallic ions(e.g., ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, and ²⁰¹Tl), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase and others commonly usedin an ELISA), electron transfer agents (e.g., including metal bindingproteins and compounds), luminescent and chemiluminescent labels (e.g.,luciferin and 2,3-dihydrophtahlazinediones, e.g., luminol), magneticbeads (e.g., DYNABEADS™), and colorimetric labels such as colloidal goldor colored glass or plastic beads (e.g., polystyrene, polypropylene,latex, etc.). For a review of various labeling or signal producingsystems that can be used, see e.g. U.S. Pat. No. 4,391,904.

5. Conjugation of a Therapeutic Moiety

In some examples, the anti-RSV antibodies and antigen-binding fragmentsprovided herein can be further modified by conjugation to a therapeuticmoiety. Exemplary therapeutic moieties include, but are not limited to,a cytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive metal ion (e.g., alpha-emitters). Exemplary cytotoxinor cytotoxic agents include, but are not limited to, any agent that isdetrimental to cells, such as, but not limited to, paclitaxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Exemplary therapeutic agents include, but are notlimited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),anti-mitotic agents (e.g., vincristine and vinblastine), and antivirals,such as, but not limited to, nucleoside analogs, such as zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, andribavirin; foscamet, amantadine, rimantadine, saquinavir, indinavir,ritonavir, and alpha-interferons.

In some examples, the anti-RSV antibodies and antigen-binding fragmentsprovided herein can be further modified by conjugation to a therapeuticmoiety that is a therapeutic polypeptide. Exemplary therapeuticpolypeptides include, but are not limited to, a toxin, such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; or animmunostimulatory agent, such as a cytokine, such as, but not limitedto, an interferon (e.g., IFN-α, β, γ, ω), a lymphokine, a hematopoieticgrowth factor, such as, for example, GM-CSF (granulocyte macrophagecolony stimulating factor), Interleukin-2 (IL-2), Interleukin-3 (IL-3),Interleukin-4 (IL-4), Interleukin-7 (IL-7), Interleukin-10 (IL-10),Interleukin-12 (IL-12), Interleukin-14 (IL-14), and Tumor NecrosisFactor (TNF).

6. Modifications to Improve Binding Specificity

The binding specificity of the anti-RSV antibodies and antibodyfragments provided can be altered or improved by techniques, such asphage display. Methods for phage display generally involve the use of afilamentous phage (phagemid) surface expression vector system forcloning and expressing antibody species of the library. Various phagemidcloning systems to produce combinatorial libraries have been describedby others. See, for example the preparation of combinatorial antibodylibraries on phagemids as described by Kang et al., (1991) Proc. Natl.Acad. Sci., USA, 88:4363-4366; Barbas et al., (1991) Proc. Natl. Acad.Sci. USA, 88:7978-7982; Zebedee et al., (1992) Proc. Natl. Acad. Sci.,USA, 89:3175-3179; Kang et al., (1991) Proc. Natl. Acad. Sci., USA,88:11120-11123; Barbas et al., (1992) Proc. Natl. Acad. Sci., USA,89:4457-4461; and Gram et al., (1992) Proc. Natl. Acad Sci., USA,89:3576-3580, which are incorporated herein by reference.

In particular examples, DNA sequences encoding V_(H) and V_(L) domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues). The DNA encoding the V_(H) and V_(L)domains are recombined together with an scFv linker by PCR and clonedinto a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13 and the V_(H) and V_(L) domains are usually recombinantly fusedto either the phage gene III or gene VIII. Phage expressing anantigen-binding domain that binds to a RSV antigen, for example, RSV Fprotein, can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead.Examples of phage display methods that can be used to make theantibodies by phage display include those disclosed, for example, inBrinkman et al. (1995) J. Immunol. Methods 182:41-50; Ames et al. (1995)J. Immunol. Methods 184:177-186; Kettleborough et al. (1994) Eur. J.Immunol. 24:952-958; Persic et al. (1997) Gene 187:9-18; Burton et al.(1994) Advances in Immunology 57:191-280; PCT publication Nos. WO90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen-binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described herein. Techniques to recombinantly produceFab, Fab′ and F(ab′)₂ fragments can also be employed using methods knownin the art such as those disclosed in PCT publication No. WO 92/22324;Mullinax et al. (1992) BioTechniques 12(6):864-869; Sawai et al. (1995)AJRI 34:26-34; and Better et al. (1988) Science 240: 1041-1043.

The resulting phagemid library can be manipulated to increase and/oralter the immunospecificities of the antibodies or antigen-bindingfragments to produce and subsequently identify additional antibodieswith improved properties, such as increased binding to a target antigen.For example, either or both the heavy and light chain encoding DNA canbe mutagenized in a complementarity determining region (CDR) of thevariable region of the immunoglobulin polypeptide, and subsequentlyscreened for desirable immunoreaction and neutralization capabilities.The resulting antibodies can then be screened in one or more of theassays described herein for determining neutralization capacity.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, human or chimeric antibodies are used.Completely human antibodies are particularly desirable for therapeutictreatment of human subjects. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences or synthetic sequences homologous to human immunoglobulinsequences. See U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

E. METHODS OF ISOLATING ANTI-RSV ANTIBODIES

Anti-RSV antibodies or antigen-binding fragments thereof can beidentified and isolated by a variety of techniques well-known in the artincluding, but not limited to, murine hybridomas (see, e.g., Olsson andKaplan (1980) Proc Natl Acad Sci USA 77:5429-5431; such antibodies canbe humanized as described elsewhere herein for use in humans),transgenic mice expressing human immunoglobulin genes (see, e.g.,Kellerman and Green (2000) Curr. Opin Biotechnol. 13:593-597), phagedisplay (see, e.g., Mancini (2004) New Microbiol. 27:315-28), andisolation from mature human immune cells, such as B cells (see, e.g.,Banchereau and Rousset (1992) Adv Immunol. 52: 125-262, Crotty and Ahmed(2004) Semin Immunol. 16: 197-203, Carsetti (2004) Methods Mol. Biol.271: 25-35, McHeyzer-Williams and McHeyzer-Williams (2005) Annu RevImmunol. 23:487-513). In an exemplary method provided herein, the humananti-RSV antibodies and antigen-binding fragments thereof providedherein are identified and isolated from human B cells.

Given the difficulty in obtaining stable hybridomas from human antibodysecreting cells, an exemplary method that has been extensively used toproduce and isolate human antibody-secreting cells is theimmortalization of human B cells with Epstein Barr Virus (EBV), which isalso known to induce polyclonal B cell activation and proliferation(see, e.g., Sugimoto et al. (2004) Cancer Res. 64:3361-3364; Bishop andBusch (2002) Microbes Infect. 4:853-857). Antibody-secreting cells havebeen produced, for example, by EBV immortalization of human B cells,such as the peripheral blood, lymph nodes, spleen, tonsils, or pleuralfluids from patients or other individuals that may be exposed to theantigen or healthy subjects pre-selected using a labeled antigen (see,e.g., Casali et al. (1986) Science 234:476-9, Yamaguchi et al. (1987)Proc Natl Acad Sci USA 84:2416-2420, Posner et al. (1991) J Immunol.146:4325-4332, Raff et al. (1988) J Exp Med. 168:905-917, Steenbakkerset al. (1993) Hum Antibod Hybrid 4:166-173, Steenbakkers et al. (1994)Mol Biol Rep. 19:125-134, Evans et al. (1988) J Immunol 140:941-943, andWallis R et al. (1989) J Clin Invest 84:214-219).

Due to the low transformability, low clonability, and the inherentinstability and heterogeneity of EBV-infected human B cells (Chan et al.(1986) J Immunol 136:106-112, and James and Bell (1987) J ImmunolMethods. 100:5-40), known techniques such as cell fusion, such as, forexample with a myeloma cell line can be employed (see, e.g., Bron et al.(1984) PNAS 81:3214-3217; Yamaguchi et al. (1987) Proc Natl Acad Sci USA84:2416-2420; Posner et al. (1991) J Immunol. 146:4325-4332, Niedbalaand Stott (1998) Hybridoma 17:299-304; Li et al. (2006) Proc Natl AcadSci USA 103:3557-62). Additional techniques for improving EBVimmortalization include, for example, immortalization with oncogenicvirus, transformation with oncogenes, mini-electrofusion, andmouse-human heterofusion in a single process (see, e.g., U.S. Pat. No.4,997,764; Steenbakkers et al. (1993) Hum Antibod Hybrid. 4:166-173;Dessain et al. (2004) J Immunol Methods. 291:109-22). Human monoclonalantibodies can be isolated from B cells that have been activated andimmortalized in the presence or in the absence of an antigen and bycombining various manipulations in cell culture as described in the art(see e.g., Borrebaeck et al. (1988) Proc Natl Acad Sci USA 85:3995-3999, Davenport et al. (1992) FEMS Microbiol Immunol. 4:335-343,Laroche-Traineau et al. (1994) Hum Antib Hybrid 5:165-177, Morgenthaleret al. (1996) J. Clin Endocrinology. 81:3155-3161, Niedbala and Kurpisz(1993) Immunol Lett. 35:93-100, Mulder et al. (1993) Hum Immunol.36:186-192, Hur et al. (2005) Cell Prolif. 38:35-45, Traggiai et al.(2004) Nat Med 10:871-875, Tsuchiyama et al. (1997) Hum Antibodies8:43-47; and PCT Pub. Nos. WO 91109115, WO 041076677, WO 88101642, WO90102795, WO 96140252, and WO 02146233).

Methods for the isolation of human antibodies from mature B cells,generally involve the isolation of a mature B cell population andscreening antibodies expressed by the B cells against a particularantigen. A variety of different populations of antibody-secreting cellscan be isolated from human donors having specific profiles (e.g. naive,vaccinated, more or less recently infected and seropositive individuals)and from different tissues (e.g. blood, tonsils, spleen, lymph nodes)where B cells reside and exert their activities (Viau and Zouali (2005)Clin Immunol. 114:17-26). In an exemplary method provided herein,anti-RSV antibodies provided herein can be isolated from a sample ofperipheral blood mononuclear cells (PBMCs), which contain B cells,isolated from human donors and/or from healthy human donors that havebeen or have a high probability of having been exposed to RSV, such ashealth care workers.

After the isolation of PBMCs from the biological samples, a specificselection of antibody-secreting cells can be performed, using one of thevarious methods described in the art, on the basis of the expression ofcell surface markers on their surface and, if appropriate, of otherproteins, as well as the proliferation activity, the metabolic and/ormorphological status of the cells. In particular, various technologiesfor the purification of antibody-secreting cells from human samples makeuse of different means and conditions for positive or negativeselection. These cells are more easily and efficiently selected byphysically separating those expressing cell surface markers specific forcells that express and secrete antibodies (e.g. human B cells). Specificprotocols are known and can be found in the literature (see, e.g.Callard and Kotowicz “Human B-cell responses to cytokines” in CytokineCell Biology: A practical Approach. Balkwill F. (ed.) Oxford UniversityPress, 2000, 17-31).

The selection of specific immune cells such as B cells, is typicallyperformed using antibodies that bind specifically to a B-cell specificcell surface protein and that can be linked to solid supports (e.g.microbeads or plastic plates) or labeled with a fluorochrome that can bedetected using fluorescence-activated cell sorting (FACS). For example,human B cells have been selected on the basis of their affinity forsupports (such as microbeads) binding CD19, CD27, and/or CD22microbeads, or for the lack of binding affinity for antibodies specificfor certain isotypes prior to EBV immortalization (see, e.g., Li et al.(1995) Biochem Biophys Res Commun 207:985-993, Bernasconi et al. (2003)Blood 101:4500-4504, and Traggiai et al. (2004) Nat Med 10:871-875). Theselection of the cell marker for purification can affect the efficiencyof the immortalization process, for example, due to intracellularsignals that are triggered by the selection process and that may altercell growth and viability. For example, CD22, which is a B-cellrestricted transmembrane protein that controls signal transductionpathways related to antigen recognition and B cell activation is anexemplary molecule for initial B cell selection. Since the CD22 positivepopulation contains cells that express antibodies having differentisotypes and specificities, other cell surface markers also can be usedfor selecting the cells, either before or after the stimulation phase.

In some examples, a specific enrichment of antibody-secreting cells canbe obtained by applying a CD27-based selection in addition to theCD22-based selection. CD27 is known to be a marker for human B cellsthat have somatically mutated variable region genes (Borst J et al.(2005) Curr Opin Immunol. 17:275-281). Additional markers such as CD5,CD24, CD25, CD86, CD38, CD45, CD70, or CD69 also can be used to eitherdeplete or enrich for the desired population of cells. Thus, dependingon factors, such as the donor's history of exposure to the antigen (e.g.an RSV antigen) and the antibody titer, total CD22-enriched B cells, orfurther enriched B cell subpopulations such as CD27 positive B cells canbe selected.

Following cell selection, and before immortalization of the cells, thepopulation of cells can be exposed to an appropriate stimulating agent.Exemplary stimulating agents include, for example, polyclonal B cellactivators, such as, but not limited to, agonists of innate immuneresponses (e.g. Toll-like receptor agonists such as CpG oligonucleotides(Bernasconi et al. (2003) Blood 101:4500-4504, Bernasconi et al. (2002)Science 298:2199-2202, Bourke et al. (2003) Blood 102:956-63; e.g. CpGnucleotides, such as for example, CpG2006, CpG2395, and CpG2395,available from Cell Sciences, Canton, Mass.) and immunomodulatorymolecules such as cytokines (e.g., interleukins known to haveimmunostimulating activities, for example, IL-2, IL-4, IL-6, IL-10, andIL-13 (see Callard and Kotowicz “Human B-cell responses to cytokines” inCytokine Cell Biology: A practical Approach. Balkwill F (ed.) OxfordUniversity Press, 2000, 17-31) and agonists of cell membrane receptorsof the TNF receptor family, in particular those activating the NF-κBpathway and proliferation in B cells, such as, but not limited to,APRIL, BAFF, CD 40 ligand (CD40L) (see, e.g., Schneider (2005) Curr OpinImmunol. 17:282-289, He et al. (2004) J Immunol. 172:3268-3279, Craxtonet al. (2003) Blood 101:4464-4471, and Tangye et al. (2003) J Immunol.170:261-269). Exemplary methods of stimulating B cells using EBVimmortalization in combination with or sequentially with one or morepolyclonal activators are known in the art (see, e.g., Traggiai et al.(2004) Nat Med 10:871-875, Tsuchiyama et al. (1997) Hum Antibodies8:43-47, Imadome et al. (2003) Proc Natl Acad Sci USA 100:7836-7840, andPCT Pub. Nos. WO 07/068758, WO 04/76677, WO 91/09115, and WO 94/24164).The combination of stimulating agents can be added to the cell culturemedium before the immortalization phase at the same time or sequentially(e.g. adding a first stimulating agent immediately after the initialcell selection and a second stimulating agent hours or days later). Thestimulating agents can be directly added in the cell culture medium fromdiluted stock solutions, or after being appropriately formulated, forexample, using liposomes or other compounds that can improve theiruptake and immunostimulatory activity (Gursel et al. (2001) J Immunol.167:3324-3328). The stimulating agents also can be attached to solidmatrices (microbeads or directly on the cell culture plates), which canallow for effective removal of the agent(s). The cells can be washedwith fresh medium one or more times and, optionally, maintained innormal cell culture medium (for example, from 1 up to 6 days) in orderto further dilute and eliminate any remaining effect of the stimulatingagents. The stimulating agent(s) also can be inhibited by addingspecific compounds into the cell culture.

The cells can be further selected on the basis of the isotype of theexpressed antibody after stimulating the cells and before exposing saidselected and stimulated cells to the immortalizing agent (i.e. betweenthe stimulation phase and the immortalization phase). The isotype-basedselection of the cells can be performed by applying means for eitherpositive (allowing the isolation of the specific cells) or negative(allowing the elimination of unwanted cells) selection. For example, apopulation of stimulated IgG positive cells can be selected positively(by FACS or magnetic cell separators) or by depleting cells that expressIgM from the population of cells, and consequently enriching for cellsthat express IgG. Separation technologies for antibody-secreting cellsusing fluorescence activated or magnetic cell separators are known inthe literature (see, e.g., Li et al. (1995) Biochem Biophys Res Commun207:985-993, Traggiai et al. (2004) Nat Med 10:871-875). Depending onthe source of antibody-secreting cells and their final use, depletion(or enrichment) of other isotype expressing cells, such as IgD or IgAexpressing cells, also can be performed. A similar approach can be usedfor isolating cells on the basis of the specific subclass, if such aprecise selection is desired (e.g., selection of human B cells thatexpress IgG1, IgG2, IgG3, or IgG4 antibodies).

Various viral immortalization agents are known in the art and can beused on antibody-secreting cells to obtain immortalizedantibody-secreting cells. Viruses that infect and immortalizeantibody-secreting cells are commonly known as lymphotropic viruses.Exemplary of such viruses are those included in the gamma class ofherpesviruses. Members of this virus family infect lymphocytes in aspecies-specific manner, and are associated with lymphoproliferativedisorders and the development of several malignancies (Nicholas (2000)J. Mol Pathol. 53:222-237 and Rickinson (2001) Philos Trans R Soc Lond BBiol Sci. 356:595-604). Exemplary viruses for use as an immortalizationagent in the methods provide include EBV (Epstein-Barr virus, also knownas herpesvirus 4), and HHV-8 (human herpesvirus 8, also known as KSHV,Kaposi's Sarcoma associated Herpesvirus), which can infect andimmortalize human lymphocytes. Other exemplary viruses for use in themethods include, but are not limited to, MHV-68 (murine herpesvirus 68),HVS (herpesvirus Samiri), RRV (Rhesus Rhadinovirus), LCV (primateLymphocryptovirus), EHV-2 (Equine Herpesvirus 2) HVA (HerpesvirusAteles), and AHV-1 (Alcelaphine Herpesvirus 1), which are otheroncogenic, lymphotropic herpesvirus having some common genetic featuresconserved amongst them and similar pathogenic effects in differentmammalian host cells.

Recombinant DNA constructs that contain specific viral proteins fromviruses employed for immortalize also have been used to immortalize Bcells (see Damania (2004) Nat Rev Microbiol. 2:656-668 and Kilger et al.(1998) EMBO J. 17:1700-1709). Similar vectors containing viral genes canbe transduced into cells in the methods provided. Methods of making suchconstructs are well-known in the art and include, for example, the useof retroviral systems or virus-like particles and packaging cell lines,which provide all the necessary factors in trans for the formation ofsuch particles.

The immortalization phase can last between one and several hours, up to2-4 days. The length of immortalization phase can be adjusted dependingof various factors such as cell viability and efficiency ofimmortalization. In some examples, the cells are immortalized with EBVfor a period of about 4 to about 24 hours. In a particular example, thecells are immortalized with EBV for a period of about 16 hours.

EBV-mediated immortalization of B cells requires the expression of thecell surface receptor CD21, which is considered as the main EBVreceptor. CD21 is present on most B cell subpopulations and regulates Bcell responses by forming a complex with CD19 and the B cell antigenreceptor (Fearon and Carroll (2000) Ann Rev Immun. 18:393-422). Theability to transform cells with EBV can be enhanced by the addition of Bcell stimulating agents, but the conditions must ensure that CD21 ismaintained on the cell surface, allowing EBV immortalization at highefficiency.

Following the immortalization phase, the immortalized cells can culturedat a low density on feeder cell layers. The feeder layer can beconstituted by irradiated non-allogenic peripheral blood cellpreparations, lymphoblastoid or fibroblast cell lines, cord bloodlymphocytes, or different types of embryonic cells. An example of a cellline having such properties is EL4-B5, mutant EL4 thymoma cell linesthat efficiently support the growth and the proliferation of B cells.Other exemplary feeder cells include irradiated B-cell depleted PMBCfeeder cells as described elsewhere herein. Growth promoting agents suchas those used to stimulate the B cell population also can be used tomaintain the immortalized B cell population following immortalization.

The immortalized populations of cells can be used for a series ofapplications, in particular related to antibody isolation,characterization and production. In some examples, DNA librariesencoding the antibodies expressed by the cells or fragments of suchantibodies can be constructed from DNA isolated from the bulk populationof cells using common recombinant techniques. In some examples asdescribed herein, the immortalized cells can be further cultured anddivided into pools of antibody-secreting cells. The pools of cells canbe cultured, for example, on feed cell layers.

In some examples, cell culture supernatants from the pools of cells arescreened in one or more rounds, for the identification of cells thatexpress antibodies having a particular antigen specificity (e.g.,antibodies that immunospecifically bind a RSV F protein). Exemplarymethods for screening antibodies and measuring binding specificity aredescribed elsewhere herein and are known in the art. Once a particularantibody is identified. DNA encoding the antibody or antigen-bindingportions thereof can be isolated from the pools of cells usingwell-known recombinant methods. As described herein. DNA isolated fromthe pools of cells can then be expressed (e.g., in a prokaryotic oreukaryotic host cell) and re-screened for the identification ofindividual clones that express the desired antibody or antigen-bindingfragment thereof. Methods of expression and purification of antibodiesis well known to one of skill in the art and is described in Section F.

In some examples as described herein, the immortalized cells can besingle cell sorted using a cell sorter (e.g., FACS), using a labeledantigen. In a particular example, cells expressing anti-RSV antibodiescan be isolated using an RSV F antigen labeled with Alexa Fluor 647 inorder to label the desired cells. Following sorting, DNA encoding theanti-RSV antibody or antigen-binding fragment thereof can then beisolated using well-known recombinant methods. DNA isolated from thepools of cells can be expressed (e.g., in a prokaryotic or eukaryotichost cell) to confirm binding to the RSV antigen.

Typically, the screening methods employed for the identification ofindividual antibodies that bind to a particular antigen result in theidentification of the antigen-binding portion of such antibodies. Togenerate full length or other derivative antibodies from theantigen-binding fragment, nucleotide sequences encoding the V_(H) and/orV_(L) chain or antigen-binding portions thereof can be isolated andcloned into vectors expressing a V_(H) constant region (e.g., the humangamma 1 constant region), V_(L) constant region (e.g., human kappa orlambda constant regions), respectively. The V_(H) and V_(L) domains alsocan be cloned into a vector expressing the selected constant regions.The heavy chain conversion vectors and light chain conversion vectorsare then co-transfected into cell lines to generate stable or transientcell lines that express full-length antibodies, e.g., IgG, usingtechniques known to those of skill in the art.

F. METHODS OF PRODUCING ANTI-RSV ANTIBODIES, AND MODIFIED OR VARIANTFORMS THEREOF AND NUCLEIC ACIDS ENCODING ANTIBODIES

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be generated by any suitable method known in the art for thepreparation of antibodies, including chemical synthesis and recombinantexpression techniques. Various combinations of host cells and vectorscan be used to receive, maintain, reproduce and amplify nucleic acids(e.g. nucleic acids encoding antibodies such as the anti-RSV antibodiesor antigen-binding fragments thereof provided), and to expresspolypeptides encoded by the nucleic acids. In general, the choice ofhost cell and vector depends on whether amplification, polypeptideexpression, and/or display on a genetic package, such as a phage, isdesired. Methods for transforming host cells are well known. Any knowntransformation method (e.g., transformation, transfection, infection,electroporation and sonoporation) can be used to transform the host cellwith nucleic acids. Procedures for the production of antibodies, such asmonoclonal antibodies and antibody fragments, such as, but not limitedto, Fab fragments and single chain antibodies are well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including, but not limited to, the use of hybridoma,recombinant expression, phage display technologies or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow et al., Antibodies: A Laboratory Manual (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling, MonoclonalAntibodies and T-Cell Hybridomas 5630681 (Elsevier N.Y. 1981).

Polypeptides, such as any set forth herein, including the anti-RSVantibodies or antigen-binding fragments thereof provided herein, can beproduced by any method known to those of skill in the art including invivo and in vitro methods. Desired polypeptides can be expressed in anorganism suitable to produce the required amounts and forms of theproteins, such as for example, needed for analysis, administration andtreatment. Expression hosts include prokaryotic and eukaryotic organismssuch as E. coli, yeast, plants, insect cells, mammalian cells, includinghuman cell lines and transgenic animals (e.g., rabbits, mice, rats, andlivestock, such as, but not limited to, goats, sheep, and cattle),including production in serum, milk and eggs. Expression hosts candiffer in their protein production levels as well as the types ofpost-translational modifications that are present on the expressedproteins. The choice of expression host can be made based on these andother factors, such as regulatory and safety considerations, productioncosts and the need and methods for purification.

1. Nucleic Acids

Provided herein are isolated nucleic acid molecules encoding an anti-RSVantibody or antigen-binding fragment thereof provided herein. In someexamples, the isolated nucleic acid molecule encodes an antibody that is30D8, 104E5, 38F10, 14G3, 90D3, 56E11, 17C9 or 69F6. In some examples,the isolated nucleic acid molecule encodes an antibody form or otherantigen-binding fragment form of 30D8, 104E5, 38F10, 14G3, 90D3, 56E11,17C9 or 69F6.

In some examples, the isolated nucleic acid molecule provided hereinencodes an antibody or antigen-binding fragment thereof containing aheavy chain having an amino acid sequence set forth in SEQ ID NOS:396,398, 400, 402, 404, 452, 454 or 456. In some examples, the isolatednucleic acid molecule provided contains a nucleic acid having a sequenceof nucleotides set forth in SEQ ID NOS:443, 445, 447, 449, 451, 477, 479or 481.

In some examples, the isolated nucleic acid molecule provided hereinencodes an antibody or antigen-binding fragment thereof containing alight chain having an amino acid sequence set forth in SEQ ID NOS:395,397, 399, 401, 403, 453, 455 or 457. In some examples, the isolatednucleic acid molecule provided contains a nucleic acid having a sequenceof nucleotides set forth in SEQ ID NOS:442, 444, 446, 448, 450, 476, 478or 480.

In some examples, the isolated nucleic acid molecule provided encodes anantibody or antigen-binding fragment thereof containing a V_(H) CDR1having an amino acid sequence set forth in SEQ ID NOS:405, 411, 417,423, 429, 437-441, 458, 464, 470 or 482-484. In some examples, theisolated nucleic acid molecule provided encodes an antibody orantigen-binding fragment thereof containing a V_(H) CDR2 having an aminoacid sequence set forth in SEQ ID NOS:406, 412, 418, 424, 430, 459, 465or 471. In some examples, the isolated nucleic acid molecule providedencodes an antibody or antigen-binding fragment thereof containing aV_(H) CDR3 having an amino acid sequence set forth in SEQ ID NOS:407,413, 419, 425, 431, 460, 466 or 472.

In some examples, the isolated nucleic acid molecule provided encodes anantibody or antigen-binding fragment thereof containing a V_(L) CDR1having an amino acid sequence set forth in SEQ ID NOS:408, 414, 420,426, 432, 461, 467 or 473. In some examples, the isolated nucleic acidmolecule provided encodes an antibody or antigen-binding fragmentthereof containing a V_(L) CDR2 having an amino acid sequence set forthin SEQ ID NOS: 409, 415, 421, 427, 433, 462, 468 or 474. In someexamples, the isolated nucleic acid molecule provided encodes anantibody or antigen-binding fragment thereof containing a V_(L) CDR3having an amino acid sequence set forth in SEQ ID NOS:410, 416, 422,428, 434, 463, 469 or 475.

Nucleic acid molecules encoding the anti-RSV antibodies orantigen-binding fragments thereof provided herein can be prepared usingwell-known recombinant techniques for manipulation of nucleic acidmolecules (see, e.g., techniques described in Sambrook et al. (1990)Molecular Cloning, A Laboratory Manual. 2d Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds. (1998)Current Protocols in Molecular Biology, John Wiley & Sons, NY). In someexamples, methods, such as, but not limited to, recombinant DNAtechniques, site directed mutagenesis, and polymerase chain reaction(PCR) can be used to generate modified antibodies or antigen-bindingfragments thereof having a different amino acid sequence, for example,to create amino acid substitutions, deletions, and/or insertions.

In some examples, one or more of the CDRs of an anti-RSV antibody orantigen-binding fragment thereof provided herein is inserted withinframework regions using routine recombinant DNA techniques. Theframework regions can be selected from naturally occurring or consensusframework regions, including human framework regions (see, e.g., Chothiaet al. (1998) J. Mol. Biol. 278: 457-479 for exemplary frameworkregions). Generally, the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody or antigen-bindingfragment thereof that maintains the antigen-binding specificity of theparent anti-RSV antibody or antigen-binding fragment thereof.Alterations to the polynucleotide can be made to improve one or moreproperties of the encoded antibody or antigen-binding fragment thereofand within the skill of the art. In some examples, one or moremodifications of the polynucleotide can be made to produce amino acidsubstitutions within the framework regions, which, for example, improvebinding of the antibody or antigen-binding fragment thereof to itsantigen. Additionally, such methods can be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds.

2. Vectors

Provided herein are vectors that contain nucleic acid or nucleic acids(encoding the heavy and light chain) encoding the anti-RSV antibodies orantigen-binding fragments thereof. Generally, nucleic acid encoding theheavy chain of an antibody is cloned into a vector and the nucleic acidencoding the light chain of an antibody is cloned into the vector. Thegenes can be cloned into a single vector for dual expression thereof, orinto separate vectors. If desired, the vectors also can contain furthersequences encoding additional constant region(s) or hinge regions togenerate other antibody forms.

Many expression vectors are available and known to those of skill in theart and can be used for expression of polypeptides. The choice ofexpression vector will be influenced by the choice of host expressionsystem. Such selection is well within the level of skill of the skilledartisan. In general, expression vectors can include transcriptionalpromoters and optionally enhancers, translational signals, andtranscriptional and translational termination signals. Expressionvectors that are used for stable transformation typically have aselectable marker which allows selection and maintenance of thetransformed cells. In some cases, an origin of replication can be usedto amplify the copy number of the vector in the cells.

Vectors also can contain additional nucleotide sequences operably linkedto the ligated nucleic acid molecule, such as, for example, an epitopetag such as for localization, e.g. a hexa-his tag or a myc tag, or a tagfor purification, for example, a GST fusion, and a sequence fordirecting protein secretion and/or membrane association.

Expression of the antibodies or antigen-binding fragments thereof can becontrolled by any promoter/enhancer known in the art. Suitable bacterialpromoters are well known in the art and described herein below. Othersuitable promoters for mammalian cells, yeast cells and insect cells arewell known in the art and some are exemplified below. Selection of thepromoter used to direct expression of a heterologous nucleic aciddepends on the particular application and is within the level of skillof the skilled artisan. Promoters which can be used include but are notlimited to eukaryotic expression vectors containing the SV40 earlypromoter (Bernoist and Chambon, (1981) Nature 290:304-310), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al. (1980) Cell 22:787-797), the herpes thymidine kinase promoter(Wagner et al., (1981) Proc. Natl. Acad Sci. USA 78:1441-1445), theregulatory sequences of the metallothionein gene (Brinster et al.,(1982) Nature 296:39-42); prokaryotic expression vectors such as theβ-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad Sci. USA78:5543) or the tac promoter (DeBoer et al., (1983) Proc. Natl. Acad.Sci. USA 80:21-25); see also “Useful Proteins from RecombinantBacteria”: in Scientific American 242:79-94 (1980)); plant expressionvectors containing the nopaline synthetase promoter (Herrera-Estrella etal., (1984) Nature 303:209-213) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al., (1981) Nucleic Acids Res. 9:2871), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., (1984) Nature 310:115-120); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., (1984) Cell 38:639-646; Ornitz etal., (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,(1987) Hepatology 7:425-515); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al., (1985) Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., (1984) Cell 38:647-658; Adams et al.,(1985) Nature 318:533-538; Alexander et al., (1987) Mol. Cell Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., (1986)Cell 45:485-495), albumin gene control region which is active in liver(Pinckert et al., (1987) Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., (1985)Mol. Cell. Biol. 5:1639-403); Hammer et al., (1987) Science 235:53-58),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al, (1987) Genes and Devel. 1:161-171), beta globin gene controlregion which is active in myeloid cells (Magram et al., (1985) Nature315:338-340); Kollias et al., (1986) Cell 46:89-94), myelin basicprotein gene control region which is active in oligodendrocyte cells ofthe brain (Readhead et al., (1987) Cell 48:703-712), myosin lightchain-2 gene control region which is active in skeletal muscle (Shani(1985) Nature 314:283-286), and gonadotrophic releasing hormone genecontrol region which is active in gonadotrophs of the hypothalamus(Mason et al., (1986) Science 234:1372-1378).

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the antibody, orportion thereof, in host cells. A typical expression cassette contains apromoter operably linked to the nucleic acid sequence encoding theantibody chain and signals required for efficient polyadenylation of thetranscript, ribosome binding sites and translation termination.Additional elements of the cassette can include enhancers. In addition,the cassette typically contains a transcription termination regiondownstream of the structural gene to provide for efficient termination.The termination region can be obtained from the same gene as thepromoter sequence or can be obtained from different genes.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with anucleic acid sequence encoding a germline antibody chain under thedirection of the polyhedron promoter or other strong baculoviruspromoter.

For purposes herein, vectors are provided that contain a sequence ofnucleotides that encodes the heavy chain and/or light chain variableregion of an anti-RSV antibody. In some examples, vectors providedherein contain a sequence of nucleotides that encodes the constantregion of an antibody operably linked to the nucleic acid sequenceencoding the variable region of the antibody. The vector can include thesequence for one or all of a C_(H)1, C_(H)2, hinge, C_(H)3 or C_(H)4and/or C_(L). Generally, such as for expression of Fabs, the vectorcontains the sequence for a C_(H)1 or C_(L) (kappa or lambda lightchains). The sequences of constant regions or hinge regions are known toone of skill in the art (see e.g. U.S. Published Application No.20080248028) and described herein.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a nucleic acid encoding an antibody or antigen-bindingfragment thereof provided herein. These methods can include in vitrorecombinant DNA and synthetic techniques and in vivo recombinants(genetic recombination). The insertion into a cloning vector can, forexample, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. If the complementaryrestriction sites used to fragment the DNA are not present in thecloning vector, the ends of the DNA molecules can be enzymaticallymodified. Alternatively, any site desired can be produced by ligatingnucleotide sequences (linkers) onto the DNA termini; these ligatedlinkers can contain specific chemically synthesized nucleic acidsencoding restriction endonuclease recognition sequences.

Exemplary plasmid vectors useful to produce the antibodies orantigen-binding fragments provided herein contain a strong promoter,such as the HCMV immediate early enhancer/promoter or the MHC class Ipromoter, an intron to enhance processing of the transcript, such as theHCMV immediate early gene intron A, and a polyadenylation (polyA)signal, such as the late SV40 polyA signal. The plasmid can bemulticistronic to enable expression of the full-length heavy and lightchains of the antibody, a single chain Fv fragment or otherimmunoglobulin fragments.

3. Cell Expression Systems

Nucleic acids encoding the anti-RSV antibodies or antigen-bindingfragments thereof provided herein can be expressed in a suitable host.Cells containing the vectors and nucleic acids encoding the anti-RSVantibodies or antigen-binding fragments thereof provided herein areprovided. Generally, any cell type that can be engineered to expressheterologous DNA and has a secretory pathway is suitable. Expressionhosts include prokaryotic and eukaryotic organisms, such as bacterialcells (e.g. E. coli), yeast cells, fungal cells, Archae, plant cells,insect cells and animal cells including human cells. Expression hostscan differ in their protein production levels as well as the types ofpost-translational modifications that are present on the expressedproteins. Further, the choice of expression host is often related to thechoice of vector and transcription and translation elements used. Forexample, the choice of expression host is often, but not always,dependent on the choice of precursor sequence utilized. For example,many heterologous signal sequences can only be expressed in a host cellof the same species (i.e., an insect cell signal sequence is optimallyexpressed in an insect cell). In contrast, other signal sequences can beused in heterologous hosts such as for example, the human serum albumin(hHSA) signal sequence which works well in yeast, insect, or mammalianhost cells and the tissue plasminogen activator pre/pro sequence whichhas been demonstrated to be functional in insect and mammalian cells(Tan et al., (2002) Protein Eng. 15:337). The choice of expression hostcan be made based on these and other factors, such as regulatory andsafety considerations, production costs and the need and methods forpurification. Thus, the vector system must be compatible with the hostcell used.

Expression in eukaryotic hosts can include expression in yeasts such asSaccharomyces cerevisiae and Pichia pastoris, insect cells such asDrosophila cells and lepidopteran cells, plants and plant cells such astobacco, corn, rice, algae, and lemna. Eukaryotic cells for expressionalso include mammalian cells lines such as Chinese hamster ovary (CHO)cells or baby hamster kidney (BHK) cells. Eukaryotic expression hostsalso include production in transgenic animals, for example, includingproduction in serum, milk and eggs.

Recombinant molecules can be introduced into host cells via, forexample, transformation, transfection, infection, electroporation andsonoporation, so that many copies of the gene sequence are generated.Generally, standard transfection methods are used to produce bacterial,mammalian, yeast, or insect cell lines that express large quantity ofantibody chains, which is then purified using standard techniques (seee.g., Colley et al. (1989) J. Biol. Chem., 264:17619-17622; Guide toProtein Purification, in Methods in Enzymology, vol. 182 (Deutscher,ed.), 1990). Transformations of eukaryotic and prokaryotic cells areperformed according to standard techniques (see, e.g., Morrison (1977)J. Bact. 132:349-351; Clark-Curtiss and Curtiss (1983) Methods inEnzymology, 101, 347-362). For example, any of the well-known proceduresfor introducing foreign nucleotide sequences into host cells can beused. These include the use of calcium phosphate transfection,polybrene, protoplast fusion, electroporation, biolistics, liposomes,microinjection, plasma vectors, viral vectors (e.g., baculovirus,vaccinia virus, adenovirus and other viruses), and any other the otherwell known methods for introducing cloned genomic DNA, cDNA, plasmidDNA, cosmid DNA, synthetic DNA or other foreign genetic material into ahost cell.

Generally, for purposes herein, host cells are transfected with a firstvector encoding at least a VH chain or heavy chain of an anti-RSVantibody and a second vector encoding at least a VL chain or light chainof the anti-RSV antibody. Host cells also can be transfected with asingle vector encoding both the heavy and light chain or portionthereof.

In one example, nucleic acid encoding the heavy chain of an antibody isligated into a first expression vector and nucleic acid encoding thelight chain of an antibody is ligated into a second expression vector.The expression vectors can be the same or different, although generallythey are sufficiently compatible to allow comparable expression ofproteins (heavy and light chain) therefrom. The first and secondexpression vectors are generally co-transfected into host cells,typically at a 1:1 ratio. Exemplary of vectors include, but are notlimited to, pγ1HC and pκLC (Tiller et al. (2008) Journal ofImmunological Methods, 329:112-24). Other expression vectors include theL chain expression vector pAG4622 and the heavy chain expression vectorpAH4604 (Coloma et al. (1992) J. Immunol. Methods, 152:89-104). ThepAG4622 vector contains the genomic sequence encoding the C-regiondomain of the human κ L chain and the gpt selectable marker. The pAH4604vectors contain the hisD selectable marker and sequences encoding thehuman H chain γ1 C-region domain.

In another example, the heavy and light chain can be cloned into asingle vector that has expression cassettes for both the heavy and lightchain. In one example, genes encoding the heavy and light chains can becloned into the mammalian expression vector pTT5 (NRC BiotechnologyResearch). In another example, genes encoding the heavy and lightchains, or portions thereof, can be cloned into pCALM (SEQ ID NO:102).

For expression of a full-length Ig, sequences encoding theV_(H)-C_(H)I-hinge-C_(H)2-C_(H)3 can be cloned into a first expressionvector and sequences encoding the V_(L)-C_(L) domains can be cloned intoa second expression vector. To generate a Fab, sequences encoding theV_(H)-C_(H)1 can be cloned into a first expression vector and sequencesencoding the V_(L)-C_(L) domains can be cloned into a second expressionvector. For a conventional antibody, a heavy chain pairs with a lightchain and a Fab monomer is generated.

Expression can be in any cell expression system known to one of skill inthe art. Exemplary cells for expression include, but are not limited to,293FS cells, HEK293-6E cells or CHO cells. Other expression vectors andhost cells are described below. Upon expression, antibody heavy andlight chains pair by disulfide bond to form a full-length antibody orfragments thereof.

a. Prokaryotic Expression

Prokaryotes, especially E. coli, provide a system for producing largeamounts of proteins and can be used to express the provided anti-RSVantibodies or antigen-binding fragments thereof. Typically, E. coli hostcells are used for amplification and expression of the provided variantpolypeptides. Transformation of E. coli is simple and rapid techniquewell known to those of skill in the art. Expression vectors for E. colican contain inducible promoters, such promoters are useful for inducinghigh levels of protein expression and for expressing proteins thatexhibit some toxicity to the host cells. Examples of inducible promotersinclude the lac promoter, the trp promoter, the hybrid tac promoter, theT7 and SP6 RNA promoters and the temperature regulated λPL promoter.

Proteins, such as any provided herein, can be expressed in thecytoplasmic environment of E. coli. For some polypeptides, thecytoplasmic environment, can result in the formation of insolubleinclusion bodies containing aggregates of the proteins. Reducing agentssuch as dithiothreitol and β-mercaptoethanol and denaturants, such asguanidine-HCl and urea can be used to resolubilize the proteins,followed by subsequent refolding of the soluble proteins. An alternativeapproach is the expression of proteins in the periplasmic space ofbacteria which provides an oxidizing environment and chaperonin-like anddisulfide isomerases and can lead to the production of soluble protein.For example, for phage display of the proteins, the proteins areexported to the periplasm so that they can be assembled into the phage.Typically, a leader sequence is fused to the protein to be expressedwhich directs the protein to the periplasm. The leader is then removedby signal peptidases inside the periplasm. Examples ofperiplasmic-targeting leader sequences include the pelB leader from thepectate lyase gene and the leader derived from the alkaline phosphatasegene. In some cases, periplasmic expression allows leakage of theexpressed protein into the culture medium. The secretion of proteinsallows quick and simple purification from the culture supernatant.Proteins that are not secreted can be obtained from the periplasm byosmotic lysis. Similar to cytoplasmic expression, in some cases proteinscan become insoluble and denaturants and reducing agents can be used tofacilitate solubilization and refolding. Temperature of induction andgrowth also can influence expression levels and solubility, typicallytemperatures between 25° C. and 37° C. are used. Typically, bacteriaproduce non-glycosylated proteins. Thus, if proteins requireglycosylation for function, glycosylation can be added in vitro afterpurification from host cells.

b. Yeast Cells

Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe,Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are wellknown yeast expression hosts that can be used to express the anti-RSVantibodies or antigen-binding fragments thereof provided herein. Yeastcan be transformed with episomal replicating vectors or by stablechromosomal integration by homologous recombination. Typically,inducible promoters are used to regulate gene expression. Examples ofsuch promoters include GAL1, GAL7 and GAL5 and metallothioneinpromoters, such as CUP1, AOX1 or other Pichia or other yeast promoter.Expression vectors often include a selectable marker such as LEU2, TRP1,H1S3 and URA3 for selection and maintenance of the transformed DNA.Proteins expressed in yeast are often soluble. Co-expression withchaperonins such as Bip and protein disulfide isomerase can improveexpression levels and solubility. Additionally, proteins expressed inyeast can be directed for secretion using secretion signal peptidefusions such as the yeast mating type alpha-factor secretion signal fromSaccharomyces cerevisae and fusions with yeast cell surface proteinssuch as the Aga2p mating adhesion receptor or the Arxula adeninivoransglucoamylase. A protease cleavage site such as for the Kex-2 protease,can be engineered to remove the fused sequences from the expressedpolypeptides as they exit the secretion pathway. Yeast also is capableof glycosylation at Asn-X-Ser/Thr motifs.

c. Insect Cells

Insect cells, particularly using baculovirus expression, can be used toexpress the anti-RSV antibodies or antigen-binding fragments thereofprovided herein. Insect cells express high levels of protein and arecapable of most of the post-translational modifications used by highereukaryotes. Baculovirus have a restrictive host range which improves thesafety and reduces regulatory concerns of eukaryotic expression. Typicalexpression vectors use a promoter for high level expression such as thepolyhedrin promoter of baculovirus. Commonly used baculovirus systemsinclude the baculoviruses such as Autographa californica nuclearpolyhedrosis virus (AcNPV), and the Bombyx mori nuclear polyhedrosisvirus (BmNPV) and an insect cell line such as Sf9 derived fromSpodoptera frugiperda, Pseudaletia unipuncta (A7S) and Danaus plexippus(DpN1). For high-level expression, the nucleotide sequence of themolecule to be expressed is fused immediately downstream of thepolyhedrin initiation codon of the virus. To generate baculovirusrecombinants capable of expressing human antibodies, a dual-expressiontransfer, such as pAcUW51 (PharMingen) is utilized. Mammalian secretionsignals are accurately processed in insect cells and can be used tosecrete the expressed protein into the culture medium. In addition, thecell lines Pseudaletia unipuncta (A7S) and Danaus plexippus (DpN1)produce proteins with glycosylation patterns similar to mammalian cellsystems.

An alternative expression system in insect cells is the use of stablytransformed cells. Cell lines such as the Schnieder 2 (S2) and Kc cells(Drosophila melanogaster) and C7 cells (Aedes albopictus) can be usedfor expression. The Drosophila metallothionein promoter can be used toinduce high levels of expression in the presence of heavy metalinduction with cadmium or copper. Expression vectors are typicallymaintained by the use of selectable markers such as neomycin andhygromycin.

Exemplary of baculovirus vectors for expression of antibodies are thebicistronic vectors pAc-κ-Fc (Progen, Biotechnik; Catalog No. PR3001);pAc-λ-Fc (Progen; PR3003); pAc-κ-CH3 (Progen; PR3000) and pAc-λ-CH3(Progen; PR3002).

d. Mammalian Cells

Mammalian expression systems can be used to express the anti-RSVantibodies or antigen-binding fragments thereof provided herein.Expression constructs can be transferred to mammalian cells by viralinfection, such as, but not limited to adenovirus or vaccinia virus, orby direct DNA transfer such as liposomes, calcium phosphate.DEAE-dextran and by physical means, such as electroporation andmicroinjection. Expression vectors for mammalian cells typically includean mRNA cap site, a TATA box, a translational initiation sequence (Kozakconsensus sequence) and polyadenylation elements. Such vectors ofteninclude transcriptional promoter-enhancers for high-level expression,for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV)promoter and the long terminal repeat of Rous sarcoma virus. Thesepromoter-enhancers are active in many cell types. Tissue and cell-typepromoters and enhancer regions also can be used for expression.Exemplary promoter/enhancer regions include, but are not limited to,those from genes such as elastase I, insulin, immunoglobulin, mousemammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin,beta globin, myelin basic protein, myosin light chain 2, andgonadotropic releasing hormone gene control. Selectable markers can beused to select for and maintain cells with the expression construct.Examples of selectable marker genes include, but are not limited to,hygromycin B phosphotransferase, adenosine deaminase, xanthine-guaninephosphoribosyl transferase, aminoglycoside phosphotransferase,dihydrofolate reductase and thymidine kinase. Antibodies can be producedusing a NEO^(R)/G418 system, a dihydrofolate reductase (DHFR) system ora glutamine synthetase (GS) system. The GS system uses joint expressionvectors, such as pEE12/pEE6, to express both heavy chain and lightchain. Fusion with cell surface signaling molecules such as TCR-ζ andFc_(ε)RI-γ can direct expression of the proteins in an active state onthe cell surface.

Many cell lines are available for mammalian expression including mouse,rat human, monkey, chicken and hamster cells. Exemplary cell linesinclude, but are not limited to, CHO, Balb/3T3, BHK, HeLa, MDCK, MT2,mouse NS0 (nonsecreting) and other myeloma cell lines, hybridoma andheterohybridoma cell lines, lymphocytes, fibroblasts, Sp2/0, COS,NIH3T3, HEK293, W138, BT483, HS578T, HTB2, BT20, T47D, 293S, 2B8, andHKB cells. Cell lines also are available adapted to serum-free mediawhich facilitates purification of secreted proteins from the cellculture media. One such example is the serum free EBNA-1 cell line (Phamet al. (2003) Biotechnol. Bioeng. 84:332-42.)

Exemplary mammalian vectors for expression of antibodies include pTT5(NRC Biotechnology Research Institute) and pCALM (set forth in SEQ IDNO:102).

e. Plants

Transgenic plant cells and plants can be to express polypeptides such asany described herein. Expression constructs are typically transferred toplants using direct DNA transfer such as microprojectile bombardment andPEG-mediated transfer into protoplasts, and with agrobacterium-mediatedtransformation. Expression vectors can include promoter and enhancersequences, transcriptional termination elements and translationalcontrol elements. Expression vectors and transformation techniques areusually divided between dicot hosts, such as Arabidopsis and tobacco,and monocot hosts, such as corn and rice. Examples of plant promotersused for expression include the cauliflower mosaic virus promoter, thenopaline synthase promoter, the ribose bisphosphate carboxylase promoterand the ubiquitin and UBQ3 promoters. Selectable markers such ashygromycin, phosphomannose isomerase and neomycin phosphotransferase areoften used to facilitate selection and maintenance of transformed cells.Transformed plant cells can be maintained in culture as cells,aggregates (callus tissue) or regenerated into whole plants. Transgenicplant cells also can include algae engineered to produce proteases ormodified proteases (see for example, Mayfield et al. (2003) Proc AcadSci USA 100:438-442). Because plants have different glycosylationpatterns than mammalian cells, this can influence the choice of proteinproduced in these hosts.

4. Purification of Antibodies

Methods for purification of polypeptides, including the anti-RSVantibodies or antigen-binding fragments thereof provided herein, fromhost cells will depend on the chosen host cells and expression systems.For secreted molecules, proteins generally are purified from the culturemedia after removing the cells. For intracellular expression, cells canbe lysed and the proteins purified from the extract. In one example,polypeptides are isolated from the host cells by centrifugation and celllysis (e.g. by repeated freeze-thaw in a dry ice/ethanol bath), followedby centrifugation and retention of the supernatant containing thepolypeptides. When transgenic organisms such as transgenic plants andanimals are used for expression, tissues or organs can be used asstarting material to make a lysed cell extract. Additionally, transgenicanimal production can include the production of polypeptides in milk oreggs, which can be collected, and if necessary further the proteins canbe extracted and further purified using standard methods in the art.

Proteins, such as the anti-RSV antibodies or antigen-binding fragmentsthereof provided herein, can be purified, for example, from lysed cellextracts, using standard protein purification techniques known in theart including but not limited to, SDS-PAGE, size fraction and sizeexclusion chromatography, ammonium sulfate precipitation and ionicexchange chromatography, such as anion exchange. Affinity purificationtechniques also can be utilized to improve the efficiency and purity ofthe preparations. For example, antibodies, receptors and other moleculesthat bind proteases can be used in affinity purification. Expressionconstructs also can be engineered to add an affinity tag to a proteinsuch as a myc epitope, GST fusion or His₆ and affinity purified with mycantibody, glutathione resin and Ni-resin, respectively. Purity can beassessed by any method known in the art including gel electrophoresisand staining and spectrophotometric techniques.

Typically, antibodies and portions thereof are purified by any procedureknown to one of skill in the art. The antibodies can be purified tosubstantial purity using standard protein purification techniques knownin the art including but not limited to, SDS-PAGE, size fraction andsize exclusion chromatography, ammonium sulfate precipitation, chelatechromatography, ionic exchange chromatography or column chromatography.For example, antibodies can be purified by column chromatography.Exemplary of a method to purify antibodies is by using columnchromatography, wherein a solid support column material is linked toProtein G, a cell surface-associated protein from Streptococcus, thatbinds immunoglobulins with high affinity. The antibodies can be purifiedto 60%, 70%, 80% purity and typically at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% purity. Purity can be assessed by standardmethods such as by SDS-PAGE and coomassie staining.

The isolated polypeptides then can be analyzed, for example, byseparation on a gel (e.g. SDS-Page gel), size fractionation (e.g.separation on a Sephacryl™ S-200 HiPrep™ 16×60 size exclusion column(Amersham from GE Healthcare Life Sciences, Piscataway, N.J.). Isolatedpolypeptides also can be analyzed in binding assays, typically bindingassays using a binding partner bound to a solid support, for example, toa plate (e.g. ELISA-based binding assays) or a bead, to determine theirability to bind desired binding partners. The binding assays describedin the sections below, which are used to assess binding of precipitatedphage displaying the polypeptides, also can be used to assesspolypeptides isolated directly from host cell lysates. For example,binding assays can be carried out to determine whether antibodypolypeptides bind to one or more antigens, for example, by coating theantigen on a solid support, such as a well of an assay plate andincubating the isolated polypeptides on the solid support, followed bywashing and detection with secondary reagents, e.g. enzyme-labeledantibodies and substrates.

G. ASSESSING ANTI-RSV ANTIBODY PROPERTIES AND ACTIVITIES

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be characterized in a variety of ways well-known to one ofskill in the art. For example, the anti-RSV antibodies orantigen-binding fragments thereof provided herein can be assayed for theability to immunospecifically bind to an F protein of human RSV. Suchassays can be performed, for example, in solution (e.g., Houghten (1992)Bio/Techniques 13:412-421), on beads (Lam (1991) Nature 354:82-84), onchips (Fodor (1993) Nature 364:555-556), on bacteria (U.S. Pat. No.5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), on plasmids (Cull et al. (1992) Proc. Natl. Acad Sci. USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. USA 87:6378-6382; and Felici (1991) J. Mol. Biol.222:301-310). Antibodies or antigen-binding fragments thereof that havebeen identified to immunospecifically bind to a RSV antigen or afragment thereof also can be assayed for their specificity and affinityfor a RSV antigen. The binding specificity, or epitope, can bedetermined, for example, by competition assays with other anti-RSVantibodies and/or virus neutralization assays using MonoclonalAntibody-Resistant Mutants (MARMs). In addition, in vitro assays and invivo animal models using the anti-RSV antibodies or antigen-bindingfragments thereof provided herein can be employed for measuring thelevel of RSV neutralization effected by contact or administration of theanti-RSV antibodies or antigen-binding fragments thereof.

1. Binding Assays

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be assessed for their ability to bind a selected target(e.g., RSV virus or isolated RSV F protein) and the specificity for suchtargets by any method known to one of skill in the art. Exemplary assaysare provided in the Examples and described herein below. Binding assayscan be performed in solution, suspension or on a solid support. Forexample, target antigens can be immobilized to a solid support (e.g. acarbon or plastic surface, a tissue culture dish or chip) and contactedwith antibody or antigen-binding fragment thereof. Unbound antibody ortarget protein can be washed away and bound complexes can then bedetected. Binding assays can be performed under conditions to reducenonspecific binding, such as by using a high ionic strength buffer(e.g., 0.3-0.4 M NaCl) with nonionic detergent (e.g. 0.1% TRITON X®-100or TWEEN® 20) and/or blocking proteins (e.g. bovine serum albumin orgelatin). Negative controls also can be included in such assays as ameasure of background binding. Binding affinities can be determinedusing Scatchard analysis (Munson et al., (1980) Anal. Biochem.,107:220), surface plasmon resonance, isothermal calorimetry, or othermethods known to one of skill in the art.

Exemplary immunoassays which can be used to analyze immunospecificbinding and cross-reactivity include, but are not limited to,competitive and non-competitive assay systems using techniques such as,but not limited to, western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), Meso Scale Discovery (MSD, Gaithersburg,Md.), “sandwich” immunoassays, immunoprecipitation assays, ELISPOT,precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays. Such assays are routine and well known in the art (see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons. Inc., New York, which is incorporated byreference herein in its entirety). Other assay formats include liposomeimmunoassays (LIA), which use liposomes designed to bind specificmolecules (e.g., antibodies) and release encapsulated reagents ormarkers. The released chemicals are then detected according to standardtechniques (see Monroe et al., (1986) Amer. Clin. Prod. Rev. 5:34-41).Exemplary immunoassays not intended by way of limitation are describedbriefly below.

Immunoprecipitation protocols generally involve lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or TRITON® X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody or antigen-binding fragment thereof of interest tothe cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at40° C., adding protein A and/or protein G sepharose beads to the celllysate, incubating for about an hour or more at 40° C., washing thebeads in lysis buffer and resuspending the beads in SDS/sample buffer.The ability of the antibody or antigen-binding fragment thereof ofinterest to immunoprecipitate a particular antigen can be assessed by,e.g., western blot analysis. One of skill in the art is knowledgeable asto the parameters that can be modified to increase the binding of theantibody or antigen-binding fragment thereof to an antigen and decreasethe background (e.g., pre-clearing the cell lysate with sepharosebeads). For further discussion regarding immunoprecipitation protocolssee, e.g., Ausubel et al., eds, 1994, Current Protocols in MolecularBiology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally involves preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody or antigen-binding fragment thereof (i.e., theantibody or antigen-binding fragment thereof of interest) diluted inblocking buffer, washing the membrane in washing buffer, blocking themembrane with a secondary antibody (which recognizes the primaryantibody, e.g., an anti-human antibody) conjugated to an enzymaticsubstrate (e.g., horseradish peroxidase or alkaline phosphatase) orradioactive molecule (e.g., ³²P or ¹²⁵I) diluted in blocking buffer,washing the membrane in wash buffer, and detecting the presence of theantigen. One of skill in the art is knowledgeable as to the parametersthat can be modified to increase the signal detected and to reduce thebackground noise. For further discussion regarding western blotprotocols see, e.g., Ausubel et al, eds, 1994, Current Protocols inMolecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs involve preparing antigen, coating the well of a 96-wellmicrotiter plate with the antigen, adding the antibody orantigen-binding fragment thereof of interest conjugated to a detectablecompound such as an enzymatic substrate (e.g., horseradish peroxidase oralkaline phosphatase) to the well and incubating for a period of time,and detecting the presence of the antigen. In ELISAs, the antibody orantigen-binding fragment thereof of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundcan be added to the well. Further, instead of coating the well with theantigen, the antibody can be coated to the well. In this case, a secondantibody conjugated to a detectable compound can be added following theaddition of the antigen of interest to the coated well. One of skill inthe art is knowledgeable as to the parameters that can be modified toincrease the signal detected as well as other variations of ELISAs knownin the art. For further discussion regarding ELISAs see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons. Inc., New York at 11.2.1. Examples 5 and 8 exemplify abinding assay for binding of anti-RSV antibodies to RSV F protein.

The binding affinity of an antibody or antigen-binding fragment thereofto an antigen and the off-rate of an antibody-antigen interaction can bedetermined, for example, by competitive binding assays. One example of acompetitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., ³H or ¹²⁵I) with the antibody orantigen-binding fragment thereof of interest in the presence ofincreasing amounts of unlabeled antigen, and the detection of theantibody or antigen-binding fragment thereof bound to the labeledantigen. The affinity of an anti-RSV antibody or antigen-bindingfragment thereof provided herein for a RSV antigen and the bindingoff-rates can be determined from the data by Scatchard plot analysis.Competition with a second antibody can also be determined usingradioimmunoassays. In this case, a RSV antigen is incubated with ananti-RSV antibody or antigen-binding fragment thereof provided hereinconjugated to a labeled compound (e.g., ³H or ¹²⁵I) in the presence ofincreasing amounts of an unlabeled second antibody. In some examples,surface plasmon resonance (e.g., BiaCore 2000, Biacore AB, Upsala,Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc.Trans. 27:335) kinetic analysis can be used to determine the binding onand off rates of antibodies or antigen-binding fragments thereof to aRSV antigen. Surface plasmon resonance kinetic analysis involvesanalyzing the binding and dissociation of a RSV antigen from chips withimmobilized antibodies or fragments thereof on their surface.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein also can be assayed for their ability to inhibit the binding ofRSV to its host cell receptor using techniques known to those of skillin the art. For example, cells expressing the receptor for RSV can becontacted with RSV in the presence or absence of an antibody orantigen-binding fragment thereof and the ability of the antibody orfragment thereof to inhibit RSV's binding can measured by, for example,flow cytometry or a scintillation assay. RSV (e.g., a RSV antigen suchas F glycoprotein or G glycoprotein) or the antibody or antibodyfragment can be labeled with a detectable compound such as a radioactivelabel (e.g., ³²P, ³⁵S, and ¹²⁵I) or a fluorescent label (e.g.,fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin,allophycocyanin, o-phthaldehyde and fluorescamine) to enable detectionof an interaction between RSV and its host cell receptor.

The ability of antibodies or antigen-binding fragments thereof toinhibit RSV from binding to its receptor also can be determined incell-free assays. For example, RSV or a RSV antigen such as Fglycoprotein can be contacted with an antibody or fragment thereof andthe ability of the antibody or antibody fragment to inhibit RSV or theRSV antigen from binding to its host cell receptor can be determined. Insome examples, the antibody or the antigen-binding fragment isimmobilized on a solid support and RSV or a RSV antigen is labeled witha detectable compound. In some examples, RSV or a RSV antigen isimmobilized on a solid support and the antibody or fragment thereof islabeled with a detectable compound. The RSV or RSV antigen can bepartially or completely purified (e.g., partially or completely free ofother polypeptides) or part of a cell lysate. In some examples, a RSVantigen can be a fusion protein comprising the RSV antigen and a domainsuch as glutathionine-S-transferase. In some examples, a RSV antigen canbe biotinylated using techniques well known to those of skill in the art(e.g., biotinylation kit, Pierce Chemicals; Rockford, Ill.).

2. Binding Specificity

The binding specificity, or epitope, of the anti-RSV antibodies orantigen binding fragments thereof provided herein can be determined byany assay known to one of skill in the art, including, but not limitedto surface plasmon resonance assays, competition assays and virusneutralization assays using Monoclonal Antibody-Resistant Mutants(MARMs). The epitope can be in the isolated protein, i.e., the isolatedF protein, or in the protein in the virus. The ability of two antibodiesto bind to the same epitope can be determined by known assays in the artsuch as, for example, surface plasmon resonance assays and antibodycompetition assays. Typically, antibodies that immunospecifically bindto the same epitope can compete for binding to the epitope, which can bemeasured, for example, by an in vitro binding competition assay (e.g.competition ELISA), using techniques known the art. Typically, a firstantibody that immunospecifically binds to the same epitope as a secondantibody can compete for binding to the epitope by about or 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, wherethe percentage competition is measured ability of the second antibody todisplace binding of the first antibody to the epitope. In exemplarycompetition assays, the antigen is incubated in the presence apredetermined limiting dilution of a labeled antibody (e.g., 50-70%saturation concentration), and serial dilutions of an unlabeledcompeting antibody. Competition is determined by measuring the bindingof the labeled antibody to the antigen for any decreases in binding inthe presence of the competing antibody. Variations of such assays,including various labeling techniques and detection methods including,for example, radiometric, fluorescent, enzymatic and colorimetricdetection, are known in the art. For example, as is exemplified inExample 10 below, antibody 30D8 and motavizumab do not compete forbinding to RSV F protein, thus indicating that antibody 30D8 binds adifferent epitope than motavizumab.

The ability of a first antibody to bind to the same epitope as a secondantibody also can be determined, for example, by virus neutralizationassays using Monoclonal Antibody-Resistant Mutants. A MARM is a mutantRSV that not neutralized by a monoclonal antibody that neutralizes thewildtype RSV virus, i.e., a MARM is an RSV escape mutant. MARMs aregenerated by culturing wildtype RSV in the presence of a monoclonalantibody for successive rounds of viral replication in the presence ofthe antibody such that after each successive round of virus replication,cytopathic effects (CPE) are observed in the presence of increasingconcentrations of antibodies until a mutant virus results that is notneutralized by the antibody. If a first antibody can neutralize a MARMgenerated against a second antibody, one can conclude that theantibodies specifically bind to or interact with different epitopes. Forexample, where a first anti-RSV antibody neutralizes wild-type RSV butnot a particular mutant RSV (i.e., MARM), a second antibody thatneutralizes the wild-type RSV but not the particular mutant RSVgenerally binds the same epitope on RSV as the first antibody. Where afirst anti-RSV antibody neutralizes wild-type RSV but not a particularmutant RSV, a second antibody that neutralizes the wild-type RSV and theparticular mutant RSV generally does not bind the same epitope on RSV asthe first antibody.

For example, as is exemplified in Example 9 below, 58c5 provided hereinis capable of neutralizing MARMs previously generated against variousanti-RSV antibodies, including MARM 1129, generated against MAb 1129,the parental antibody to palivizumab and motavizumab (see, Johnson etal. (1997) J. Infect. Diseases 176:1215-1224 and U.S. Pat. No.5,824,307), MARM 19, generated against Fab 19 (see Barbas et al. (1992)Proc. Natl. Acad. Sci. USA 89:10164-10168) and MARM 151, generatedagainst MAb 151 (see, Mufson et al., (1985) J. Gen. Virol,66:2111-2124). Thus, 58c5 binds a different epitope on the F proteinthen antibodies Fab 19, MAb 151 and MAb 1129.

As is exemplified in Example 11 below, MARMs were generated againstmotavizumab and the IgG form of 58c5. The motavizumab MARM, generatedafter 5-7 rounds of selection, contains a single amino acid mutation(K272E, SEQ ID NO:486) compared to the wildtype RSV F protein. Mutationat amino acid K272 is consistant with known mutations that disruptbinding of the parent antibody of motavizumab (see, Zhao et al., (2004)J. Infectious Disease 190:1941-1946). The 58c5 MARM, generated after 10rounds of selection, contains 3 amino acid mutations (N63K, M115K andE295G, SEQ ID NO:487) compared to the wildtype RSV F protein. Themutations effecting escape in the 58c5 MARM have not been previouslyidentified as antigenic sites for various monoclonal antibodies thatimmunospecifically bind to the RSV F protein (see, e.g., Beeler et al.(1989) J. Virology 63(7):2841-2950, Crowe et al. (1998) Virology252:373-375; Zhao et al., (2004) J. Infectious Disease 190:1941-1946;Liu et al., (2007) Virology Journal 4:71). After 12 rounds of viralreplication in the presence of the IgG form of 30D8, the RSV virus wasunable to escape neutralization (see Example 11 below). The ability ofantibody 30D8 to limit the generation of MARMs means that 30D8 binds toan epitope that is less susceptible to generation of escape mutants, andas a result, antibody 30D8 is useful as a therapeutic antibody for thetreatment of a RSV infection.

Additionally, as is shown in Example 11 below, 30D8 neutralizes the boththe motavizumab MARM and the 58c5 MARM, 58c5 neutralizes the motavizumabMARM, and motavizumab neutralizes the 58c5 MARM. Thus, 30D8 binds adifferent epitope of the RSV F protein than 58c5 and motavizumab.Additionally, 58c5 binds a different epitope of the RSV F protein than30D8 and motavizumab.

3. In Vitro Assays for Analyzing Virus Neutralization Effects ofAntibodies

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be analyzed by any suitable method known in the art for thedetection of viral neutralization. Methods for detection of viralneutralization include, but are not limited to, plaque assays and assaysfor inhibition of syncytium formation. Such assays can be employed toassess, for example, inhibition of viral attachment, viral entry andcell-to-cell spread of the virus (see, e.g. Burioni et al., (1994) Proc.Natl. Acad. Sci. U.S.A. 91:355-359; Sanna et al. (2000) Virology270:386-3961; and De Logu et al., (1998) J Clin Microbiol 36:3198-3204).One of skill in the art can identify any assay capable of measuringviral neutralization.

Standard plaque assays include, for example, plaque reduction assays,plaque size reduction assays, neutralization assays and neutralizationkinetic assays. These assays measure the formation of viral plaques(i.e. areas of lysed cells) following infection of target cellmonolayers by a virus. Exemplary target cell lines that can be used inplaque reduction assays include, but are not limited to, Vero cells,MRC-5 cells, RC-37 cells, BHK-21/C13 cells and HEp-2 cells. One of skillin the art can identify appropriate target cell lines for use in aplaque assay. Selection of an appropriate cell line for a plaque assaycan depend on known factors, such as, for example, cell infectivity andthe ability of the virus to propagate in and lyse the target cell.Examples 6 and 9 exemplify in vitro neutralization assays.

Plaque reduction assays can be used to measure the ability of theanti-RSV antibody or antigen-binding fragment thereof to effect viralneutralization in solution. In exemplary plaque reduction assays, theantibody or antigen-binding fragment thereof and the virus arepre-incubated prior to the addition of target cells. Target cells arethen infected with the antibody/virus mixture and a plaque assay isperformed following a predetermined infection period. One of skill inthe art can determine the incubation times required based on knownexamples in the art. A reduction in the number of virus plaques producedfollowing infection of the target cells indicates the ability of theantibody or antigen-binding fragment thereof to prevent binding of thevirus to the target cells independent of antibody or antigen-bindingfragment thereof attachment to the target cell and/or antibody, orantigen-binding fragment thereof, internalization.

Plaque size reduction assays can be used to measure the ability of theanti-RSV antibody or antigen-binding fragment thereof to inhibit ofviral cell-to-cell spread. In exemplary plaque size reduction assays,the target cells are first infected with the virus for a predeterminedinfection period and then the antibody or antigen-binding fragmentthereof is added to the infected cell. One of skill in the art candetermine the incubation times required based on known examples in theart. A reduction in the size (i.e. diameter) of the virus plaquesindicates that the antibody or antigen-binding fragment thereof iscapable of preventing viral cell-to-cell spread.

Virus neutralization assays can be used to measure the ability of theanti-RSV antibody or antigen-binding fragment thereof to effect viralneutralization at the target cell surface by association of the antibodyor antigen-binding fragment thereof with the target cell prior to virusexposure. In exemplary virus neutralization assays, the antibody orantigen-binding fragment thereof and target cells are pre-incubated fora predetermined period of time to allow for binding of the antibody orantigen-binding fragment thereof to the targeted cell. Following thepre-incubation period, the unbound antibody is removed and the targetcells are infected with the virus. A reduction in the number of plaquesin this assay indicates the ability of the antibody or antigen-bindingfragment thereof to prevent viral infection dependent upon attachment tothe target cell and/or internalization of the antibody orantigen-binding fragment thereof. This assay also can be used to measureneutralization kinetics by varying antibody or antigen-binding fragmentconcentrations and pre-incubation times.

Exemplary assays for inhibition of syncytium formation can be employedto measure antibody-mediated inhibition of viral cytopathic effects byblocking the formation of syncytia when using a fusogenic viral strain.One of skill in the art can identify an appropriate fusogenic viralstrain for use in the assay.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein also can be assayed for their ability to inhibit or downregulateRSV replication using techniques known to those of skill in the art. Forexample, RSV replication can be assayed by a plaque assay such asdescribed, e.g., by Johnson et al. (1997) Journal of Infectious Diseases176:1215-1224. The anti-RSV antibodies or antigen-binding fragmentsthereof provided herein also can be assayed for their ability to inhibitor downregulate the expression of RSV polypeptides. Techniques known tothose of skill in the art, including, but not limited to, Western blotanalysis, Northern blot analysis, and RT-PCR can be used to measure theexpression of RSV polypeptides.

4. In Vivo Animal Models for Assessing Efficacy of the Anti-RSVAntibodies

In vivo studies using animal models can be performed to assess theefficacy of the anti-RSV antibodies or antigen-binding fragments thereofprovided herein. In vivo studies using animal models can be performed toassess any toxicity of administration of such antibodies orantigen-binding fragments thereof. A variety of assays, such as thoseemploying in vivo animal models, are available to those of skill in theart for evaluating the ability of the anti-RSV antibodies to inhibit ortreat RSV virus infection and for assaying any toxicity. The therapeuticeffect of the anti-RSV antibodies can be assessed using animal models ofthe pathogenic infection, including animal models of viral infection.Such animal models are known in the art, and include, but are notlimited to, animal models for RSV infection, such as but not limited tocotton rat, inbred mouse, calf, ferret, hamster, guinea pig, chimpanzee,owl monkey, rhesus monkey, African green monkey, cebus monkey, squirrelmonkey, bonnet monkey, baboon, (see, e.g., Prince et al. (1978) Am. J.Pathol. 93:771-791; Prince et al. (1979) Infect. Immunol. 26:764-766;Byrd and Prince (1997) Clinical Infectious Diseases 25:1363-1368,including references cited therein, for exemplary models of RSVinfection). For in vivo testing of an antibody or antigen-bindingfragment or composition's toxicity, any animal model system known in theart can be used, including, but not limited to, rats, mice, cows,monkeys, and rabbits.

5. In Vitro and In Vivo Assays for Measuring Antibody Efficacy

Efficacy in treating or preventing viral infection can be demonstratedby detecting the ability of a anti-RSV antibody or antigen-bindingfragment thereof provided herein to inhibit the replication of thevirus, to inhibit transmission or prevent the virus from establishingitself in its host, to reduce the incidence of RSV infection, or toprevent, ameliorate or alleviate one or more symptoms associated withRSV infection. The treatment is considered therapeutic if there is, forexample, a reduction is viral load, amelioration of one or moresymptoms, a reduction in the duration of a RSV infection, or a decreasein mortality and/or morbidity following administration of an antibody orcomposition provided herein. Further, the treatment is consideredtherapeutic if there is an increase in the immune response following theadministration of one or more antibodies or antigen-binding fragmentsthereof which immunospecifically bind to one or more RSV antigens.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be tested in vitro and in vivo for the ability to induce theexpression of cytokines such as IFN-α, IFN-β, IFN-γ, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL-15. Techniques knownto those of skill in the art can be used to measure the level ofexpression of cytokines. For example, the level of expression ofcytokines can be measured by analyzing the level of RNA of cytokines by,for example, RT-PCR and Northern blot analysis, and by analyzing thelevel of cytokines by, for example, immunoprecipitation followed byWestern blot analysis or ELISA.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be tested in vitro and in vivo for their ability to modulatethe biological activity of immune cells, including human immune cells(e.g., T-cells, B-cells, and Natural Killer cells). The ability of ananti-RSV antibody or antigen-binding fragment to modulate the biologicalactivity of immune cells can be assessed by detecting the expression ofantigens, detecting the proliferation of immune cells, detecting theactivation of signaling molecules, detecting the effector function ofimmune cells, or detecting the differentiation of immune cells.Techniques known to those of skill in the art can be used for measuringthese activities. For example, cellular proliferation can be assayed by³H-thymidine incorporation assays and trypan blue cell counts. Antigenexpression can be assayed, for example, by immunoassays including, butare not limited to, competitive and non-competitive assay systems usingtechniques such as western blots, immunohistochemistryradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, precipitin reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, protein A immunoassays and FACS analysis. Theactivation of signaling molecules can be assayed, for example, by kinaseassays and electrophoretic shift assays (EMSAs).

The anti-RSV antibodies or antigen-binding fragments thereof providedherein also can be tested for their ability to inhibit viral replicationor reduce viral load in in vitro, ex vivo and in vivo assays. Theanti-RSV antibodies or antigen-binding fragments thereof also can beassayed for their ability to decrease the time course of RSV infection.The anti-RSV antibodies or antigen-binding fragments thereof also can beassayed for their ability to increase the survival period of humanssuffering from RSV infection by at least or about 25%, at least or about50%, at least or about 60%, at least or about 75%, at least or about85%, at least or about 95%, or at least or about 99%. Further, anti-RSVantibodies or antigen-binding fragments thereof can be assayed for theirability reduce the hospitalization period of humans suffering from RSVinfection by at least or about 60%, at least or about 75%, at least orabout 85%, at least or about 95%, or at least or about 99%. Techniquesknown to those of skill in the art can be used to analyze the functionof the anti-RSV antibodies or antigen-binding fragments thereof providedherein in vivo.

In accordance with the methods and uses provided herein, clinical trialswith human subjects need not be performed in order to demonstrate theprophylactic and/or therapeutic efficacy of the anti-RSV antibodies orantigen-binding fragments thereof provided herein. In vitro and animalmodel studies using the anti-RSV antibodies or antigen-binding fragmentsthereof provided herein can be extrapolated to humans and are sufficientfor demonstrating the prophylactic and/or therapeutic utility of theanti-RSV antibodies or antigen-binding fragments.

H. DIAGNOSTIC USES

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be used in diagnostic assays for the detection, purification,and/or neutralization of RSV. Exemplary diagnostic assays include invitro and in vivo detection of RSV. For example, assays using theanti-RSV antibodies or antigen-binding fragments thereof provided hereinfor qualitatively and quantitatively measuring levels of RSV in anisolated biological sample (e.g., sputum) or in vivo are provided.

As described herein, the anti-RSV antibodies or antigen-bindingfragments thereof can be conjugated to a detectable moiety for in vitroor in vivo detection. Such antibodies can be employed, for example, toevaluate the localization and/or persistence of the anti-RSV antibody orantigen-binding fragment thereof at an in vivo site, such as, forexample, a mucosal site. The anti-RSV antibodies or antigen-bindingfragments thereof which are coupled to a detectable moiety can bedetected in vivo by any suitable method known in the art. The anti-RSVantibodies or antigen-binding fragments thereof which are coupled to adetectable moiety also can be detected in isolated biological samples,such as tissue or fluid samples obtained from the subject followingadministration of the antibody or antigen-binding fragment thereof.

1. In Vitro Detection of Pathogenic Infection

In general, RSV can be detected in a subject or patient based on thepresence of one or more RSV proteins and/or polynucleotides encodingsuch proteins in a biological sample (e.g., blood, sera, sputum urineand/or other appropriate cells or tissues) obtained from a subject orpatient. Such proteins can be used as markers to indicate the presenceor absence of RSV in a subject or patient. The anti-RSV antibodies orantigen-binding fragments thereof provided herein can be employed fordetection of the level of antigen and/or epitope that binds to the agentin the biological sample.

A variety of assay formats are known to those of ordinary skill in theart for using a anti-RSV antibody or antigen-binding fragment thereof todetect polypeptide markers in a sample (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In general, the presence or absence of RSV in a subject or patient canbe determined by contacting a biological sample obtained from a subjector patient with an anti-RSV antibody or antigen-binding fragment thereofprovided herein and detecting in the sample a level of polypeptide thatbinds to the anti-RSV antibody or antigen-binding fragment thereof.

In some examples, the assay involves the use of an anti-RSV antibody orantigen-binding fragment thereof provided herein immobilized on a solidsupport to bind to and remove the target polypeptide from the remainderof the sample. The bound polypeptide can then be detected using adetection reagent that contains a reporter group and specifically bindsto the antibody/polypeptide complex. Such detection reagents cancontain, for example, a binding agent that specifically binds to thepolypeptide or an antibody or other agent that specifically binds to thebinding agent.

In some examples, a competitive assay can be utilized, in which apolypeptide is labeled with a reporter group and allowed to bind to theimmobilized anti-RSV antibody or antigen-binding fragment thereof afterincubation of the anti-RSV antibody or antigen-binding fragment thereofwith the sample. The extent to which components of the sample inhibitthe binding of the labeled polypeptide to the anti-RSV antibody orantigen-binding fragment thereof is indicative of the reactivity of thesample with the immobilized anti-RSV antibody or antigen-bindingfragment thereof. Suitable polypeptides for use within such assaysinclude full length RSV F proteins and portions thereof, including theextracellular domain of a RSV F protein, to which an anti-RSV antibodyor antigen-binding fragment thereof binds, as described above.

The solid support can be any material known to those of ordinary skillin the art to which the protein can be attached. For example, the solidsupport can be a test well in a microtiter plate or a nitrocellulose orother suitable membrane. The support also can be a bead or disc, such asglass, fiberglass, latex or a plastic material such as polystyrene orpolyvinylchloride. The support also can be a magnetic particle or afiber optic sensor, such as those disclosed, for example, in U.S. Pat.No. 5,359,681. The anti-RSV antibody or antigen-binding fragment thereofcan be immobilized on the solid support using a variety of techniquesknown to those of skill in the art. The anti-RSV antibody orantigen-binding fragment thereof can be immobilized by adsorption to awell in a microtiter plate or to a membrane. In such cases, adsorptioncan be achieved by contacting the anti-RSV antibody or antigen-bindingfragment thereof, in a suitable buffer, with the solid support for asuitable amount of time. The contact time varies with temperature, butis typically between about 1 hour and about 1 day. In general,contacting a well of a plastic microtiter plate (such as polystyrene orpolyvinylchloride) with an amount of anti-RSV antibody orantigen-binding fragment thereof ranging from about 10 ng to about 10μg, and typically about 100 ng to about 1 μg, is sufficient toimmobilize an adequate amount of anti-RSV antibody or antigen-bindingfragment thereof.

Covalent attachment of anti-RSV antibody or antigen-binding fragmentthereof to a solid support can generally be achieved by first reactingthe support with a bifunctional reagent that will react with the supportand a functional group, such as a hydroxyl or amino group, on theanti-RSV antibody or antigen-binding fragment thereof. For example, theanti-RSV antibody or antigen-binding fragment thereof can be covalentlyattached to supports having an appropriate polymer coating usingbenzoquinone or by condensation of an aldehyde group on the support withan amine and an active hydrogen on the binding partner (see, e.g.,Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

In some examples, the assay is performed in a flow-through or strip testformat, wherein the anti-RSV antibody or antigen-binding fragmentthereof is immobilized on a membrane, such as nitrocellulose. In theflow-through test, polypeptides within the sample bind to theimmobilized anti-RSV antibody or antigen-binding fragment thereof as thesample passes through the membrane. A second, labeled binding agent thenbinds to the anti-RSV antibody or antigen-binding fragmentthereof-polypeptide complex as a solution containing the second bindingagent flows through the membrane.

Additional assay protocols exist in the art that are suitable for usewith the RSV proteins or anti-RSV antibodies or antigen-bindingfragments thereof provided. The above descriptions are intended to beexemplary only. For example, it will be apparent to those of ordinaryskill in the art that the above protocols can be readily modified to useRSV polypeptides to detect antibodies that bind to such polypeptides ina biological sample. The detection of such protein-specific antibodiescan allow for the identification of RSV infection.

To improve sensitivity, multiple RSV protein markers can be assayedwithin a given sample. It will be apparent that anti-RSV antibodies orantigen-binding fragments thereof specific for different RSVpolypeptides can be combined within a single assay. Further, multipleprimers or probes can be used concurrently. The selection of RSV proteinmarkers can be based on routine experiments to determine combinationsthat results in optimal sensitivity. In addition, or alternatively,assays for RSV proteins provided herein can be combined with assays forother known RSV antigens.

2. In Vivo Detection of Pathogenic Infection

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be employed as an in vivo diagnostic agent. For example, theanti-RSV antibodies or antigen-binding fragments thereof can provide animage of infected tissues (e.g., RSV infection in the lungs) usingdetection methods such as, for example, magnetic resonance imaging,X-ray imaging, computerized emission tomography and other imagingtechnologies. For the imaging of RSV infected tissues, for example, theantibody portion of the anti-RSV antibody generally will bind to RSV(e.g., binding a RSV F protein epitope), and the imaging agent will bean agent detectable upon imaging, such as a paramagnetic, radioactive orfluorescent agent that is coupled to the anti-RSV antibody orantigen-binding fragment thereof. Generally, for use as a diagnosticagent, the anti-RSV antibody or antigen-binding fragment thereof iscoupled directly or indirectly to the imaging agent.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to the anti-RSV antibodies or antigen-binding fragments(see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509). Exemplaryattachment methods involve the use of a metal chelate complex employing,for example, an organic chelating agent such a DTPA attached to theantibody or antigen-binding fragment thereof (U.S. Pat. No. 4,472,509).The antibodies also can be reacted with an enzyme in the presence of acoupling agent such as glutaraldehyde or periodate. Conjugates withfluorescein markers are prepared in the presence of such coupling agentsor by reaction with an isothiocyanate.

For in vivo diagnostic imaging, the type of detection instrumentavailable is considered when selecting a given radioisotope. Theradioisotope selected has a type of decay which is detectable for agiven type of instrument. Another factor in selecting a radioisotope forin vivo diagnosis is that the half-life of the radioisotope be longenough so that it is still detectable at the time of maximum uptake bythe target, but short enough so that deleterious radiation with respectto the host is minimized. Typically, a radioisotope used for in vivoimaging will lack a particle emission, but produce a large number ofphotons in the 140-250 keV range, which can be readily detected byconventional gamma cameras.

For in vivo diagnosis, radioisotopes can be bound to the antibodies orantigen-binding fragments thereof provided herein either directly orindirectly by using an intermediate functional group. Exemplaryintermediate functional groups which can be used to bind radioisotopes,which exist as metallic ions, to antibodies include bifunctionalchelating agents, such as diethylene-triamine pentaacetic acid (DTPA)and ethylenediaminetetraacetic acid (EDTA) and similar molecules.Examples of metallic ions which can be bound to the anti-RSV antibodiesor antigen-binding fragments thereof provided include, but are notlimited to, ⁷²Arsenic, ²¹¹Astatine, ¹⁴Carbon, ⁵¹Chromium, ³⁶Chlorine,⁵⁷Cobalt, ⁵⁸Cobalt, ⁶⁷Copper, ¹⁵²Europium, ⁶⁷Gallium, ⁶⁸Gallium,³Hydrogen, ¹²³Iodine, ¹²⁵Iodine, ¹³¹Iodine, ¹¹¹Indium, ⁵⁹Iron,³²Phosphorus, ¹⁸⁶Rhenium, ¹⁸⁸ Rhenium, ⁹⁷Ruthenium, ⁷⁵Selenium,³⁵Sulphur, ^(99m)Technicium, ²⁰¹Thalium, ⁹⁰Yttrium and ⁸⁹Zirconium.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be labeled with a paramagnetic isotope for purposes of invivo diagnosis, as in magnetic resonance imaging (MRI) or electron spinresonance (ESR). In general, any conventional method for visualizingdiagnostic imaging can be utilized. Generally, gamma and positronemitting radioisotopes are used for camera imaging and paramagneticisotopes for MRI. Elements which are particularly useful in suchtechniques include, but are not limited to, ⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and⁵⁶Fe.

Exemplary paramagnetic ions include, but are not limited to, chromium(III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II),copper (II), neodymium (III), samarium (III), ytterbium (III),gadolinium (III), vanadium (II), terbium (III), dysprosium (III),holmium (III) and erbium (III). Ions useful, for example, in X-rayimaging, include but are not limited to lanthanum (III), gold (III),lead (II), and bismuth (III).

The concentration of detectably labeled anti-RSV antibody orantigen-binding fragment thereof which is administered is sufficientsuch that the binding to RSV is detectable compared to the background.Further, it is desirable that the detectably labeled anti-RSV antibodyor antigen-binding fragment thereof be rapidly cleared from thecirculatory system in order to give the best target-to-background signalratio.

The dosage of detectably labeled anti-RSV antibody or antigen-bindingfragment thereof for in vivo diagnosis will vary depending on suchfactors as age, sex, and extent of disease of the individual. The dosageof a human monoclonal antibody can vary, for example, from about 0.01mg/m² to about 500 mg/m², 0.1 mg/m² to about 200 mg/m², or about 0.1mg/m² to about 10 mg/m². Such dosages can vary, for example, dependingon whether multiple injections are given, tissue, and other factorsknown to those of skill in the art.

3. Monitoring Infection

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be used in vitro and in vivo to monitor the course ofpathogenic disease therapy. Thus, for example, the increase or decreasein the number of cells infected with RSV or changes in the concentrationof the RSV virus particles present in the body or in various body fluidscan be measured. Using such methods, the anti-RSV antibodies orantigen-binding fragments thereof can be employed to determine whether aparticular therapeutic regimen aimed at ameliorating the pathogenicdisease is effective.

I. PROPHYLACTIC AND THERAPEUTIC USES

The anti-RSV antibodies or antigen-binding fragments thereof providedherein and pharmaceutical compositions containing anti-RSV antibodies orantigen-binding fragments thereof provided herein can be administered toa subject for prophylaxis and therapy. For example, the antibodies orantigen-binding fragments thereof provided can be administered fortreatment of a disease or condition, such as a RSV infection. In someexamples, the antibodies or antigen-binding fragments thereof providedcan be administered to a subject for prophylactic uses, such as theprevention and/or spread of RSV infection, including, but not limited tothe inhibition of establishment of RSV infection in a host or inhibitionof RSV transmission between subjects. In some examples, the antibodiesor antigen-binding fragments thereof provided can be administered to asubject for the reduction of RSV viral load in the subject. Theantibodies or antigen-binding fragments thereof also can be administeredto a subject for preventing, treating, and/or alleviating of one or moresymptoms of a RSV infection or reduce the duration of a RSV infection.

In some examples, administration of an anti-RSV antibody orantigen-binding fragment thereof provided herein inhibits the incidenceof RSV infection by at least or about 99%, at least or about 95%, atleast or about 90%, at least or about 85%, at least or about 80%, atleast or about 75%, at least or about 70%, at least or about 65%, atleast or about 60%, at least or about 55%, at least or about 50%, atleast or about 45%, at least or about 40%, at least or about 35%, atleast or about 30%, at least or about 25%, at least or about 20%, atleast or about 15%, or at least or about 10% relative to the incidenceof RSV infection in the absence of the anti-RSV antibody orantigen-binding fragment. In some examples, administration of ananti-RSV antibody or antigen-binding fragment provided herein decreasesthe severity of one or more symptoms of RSV infection by at least orabout 99%, at least or about 95%, at least or about 90%, at least orabout 85%, at least or about 80%, at least or about 75%, at least orabout 70%, at least or about 65%, at least or about 60%, at least orabout 55%, at least or about 50%, at least or about 45%, at least orabout 40%, at least or about 35%, at least or about 30%, at least orabout 25%, at least or about 20%, at least or about 15%, or at least orabout 10% relative to the severity of the one or more symptoms of RSVinfection in the absence of the anti-RSV antibody or antigen-bindingfragment.

1. Subjects for Therapy

A subject or candidate for therapy with an anti-RSV antibody orantigen-binding fragment thereof provided herein includes, but is notlimited to, a subject, such as a human patient, that has been exposed toa RSV virus, a subject, such as a human patient, who exhibits one ormore symptoms of a RSV infection and a subject, such as a human patient,who is at risk of a RSV infection. Exemplary RSV virus infectionsinclude those caused by RSV viruses, such as, but not limited to, acuteRSV disease, RSV upper respiratory tract infection (URI) and/or RSVlower respiratory tract infection (LRI), including, for example,bronchiolitis and pneumonia.

In some examples, the subject for therapy with an anti-RSV antibody orantigen-binding fragment thereof provided herein is a mammal. In someexamples, the subject for therapy with an anti-RSV antibody orantigen-binding fragment thereof provided herein is a primate. Inparticular examples, the subject for therapy with an anti-RSV antibodyor antigen-binding fragment thereof provided herein is a human.

The provided anti-RSV antibodies or antigen-binding fragments thereofcan be administered to a subject, such as a human patient, for thetreatment of any RSV-mediated disease. For example, the anti-RSVantibodies or antigen-binding fragments thereof provided herein can beadministered to a subject to alleviate one or more symptoms orconditions associated with a RSV virus infection, including, but notlimited to, asthma, wheezing, reactive airway disease (RAD), and chronicobstructive pulmonary disease (COPD). Such diseases and condition arewell known and readily diagnosed by physicians or ordinary skill.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be administered to a subject, such a human patient, having aRSV virus infection for the maintenance or suppression therapy ofrecurring RSV virus-mediated disease.

The provided anti-RSV antibodies or antigen-binding fragments thereofcan be administered to a subject, such as a human patient, at risk of aRSV virus infection, including, but not limited to, a prematurely born(pre-term) infant (e.g. a human infant born less than 38 weeks ofgestational age, such as, for example, 29 weeks, 30 weeks, 31 weeks, 32weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, or 37 weeks gestationalage); an infant (e.g., a human infant born more than 37 weeksgestational age), a subject having cystic fibrosis, bronchopulmonarydysplasia, congenital heart disease, congenital immunodeficiency, oracquired immunodeficiency (e.g., an AIDS patient), leukemia, non-Hodgkinlymphoma, an immunosuppressed patient such as, for example, a recipientof a transplant (e.g. a bone marrow transplant or a kidney transplant),or elderly subjects, including individuals in nursing homes orrehabilitation centers. In some examples, the anti-RSV antibodies orantigen-binding fragments thereof provided herein can be administered toa subject, such as a pre-term infant or infant exposed to one or moreenvironmental risk factors, such as, but not limited to attendingdaycare, having school aged siblings, exposure to environmental airpollutants, congenital airway abnormalities, and/or severe neuromusculardisease. In some examples, the provided anti-RSV antibodies orantigen-binding fragments thereof can be administered to a subject, suchan infant or child who is younger than two years, having chronic lungdisease or congenital heart disease, including congestive heart failure,pulmonary hypertension, and cyanotic heart disease.

Tests for various pathogens and pathogenic infection are known in theart and can be employed for the assessing whether a subject is acandidate for therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein. For example, tests for RSV virusinfection, are known and include for example, viral culture plaqueassays, antigen detection test, polymerase chain reaction (PCR) tests,and various antibody serological tests. Tests for viral infection can beperformed on samples obtained from tissue or fluid samples, such asspinal fluid, blood, or urine. Additional tests include, but are notlimited to chest X-rays, which can show signs of pneumonia, other bloodtests, such as a chemistry screening, a complete blood count, orarterial blood gases (ABGs) analysis, and oximetry, to measure theamount of oxygen in the blood.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be administered to a subject, who is at an increased risk ofRSV infection during particular times of the year. RSV season typicallyextends from October through May. Subjects, who exhibit increasedsusceptibility to virus infection during this time, such as infants theelderly or immunocompromised patients, can be administered an anti-RSVantibody or antigen-binding fragment thereof provided herein for theprophylaxis and/or treatment of RSV infection just prior to and/orduring RSV season. In some examples, the anti-RSV antibody orantigen-binding fragment thereof provided herein is administered onetime, two times, three times, four times or five times during RSVseason. In some examples, the anti-RSV antibody or antigen-bindingfragment thereof provided herein is administered one time, two times,three times, four times or five times within one month, two months orthree months, prior to a RSV season.

2. Dosages

The anti-RSV antibody or antigen-binding fragment thereof providedherein is administered in an amount sufficient to exert atherapeutically useful effect in the absence of undesirable side effectson the patient treated. The therapeutically effective concentration ofan anti-RSV antibody or antigen-binding fragment thereof can bedetermined empirically by testing the polypeptides in known in vitro andin vivo systems such as by using the assays provided herein or known inthe art.

An effective amount of antibody or antigen-binding fragment thereof tobe administered therapeutically will depend, for example, upon thetherapeutic objectives, the route of administration, and the conditionof the patient. In addition, the attending physician takes intoconsideration various factors known to modify the action of drugs,including severity and type of disease, patient's health, body weight,sex, diet, time and route of administration, other medications and otherrelevant clinical factors. Accordingly, it will be necessary for thetherapist to titer the dosage of the antibody or antigen-bindingfragment thereof and modify the route of administration as required toobtain the optimal therapeutic effect. Typically, the clinician willadminister the antibody or antigen-binding fragment thereof until adosage is reached that achieves the desired effect. The progress of thistherapy is easily monitored by conventional assays. Exemplary assays formonitoring treatment of a viral infection are know in the art andinclude for example, viral titer assays.

Generally, the dosage ranges for the administration of the anti-RSVantibodies or antigen-binding fragments thereof provided herein arethose large enough to produce the desired effect in which the symptom(s)of the pathogen-mediated disease (e.g. viral disease) are ameliorated orthe likelihood of virus infection is decreased. In some examples, theanti-RSV antibodies or antigen-binding fragments thereof provided hereinare administered in an amount effective for inducing an immune responsein the subject. The dosage is not so large as to cause adverse sideeffects, such as hyperviscosity syndromes, pulmonary edema or congestiveheart failure. Generally, the dosage will vary with the age, condition,sex and the extent of the disease in the patient and can be determinedby one of skill in the art. The dosage can be adjusted by the individualphysician in the event of the appearance of any adverse side effect.Exemplary dosages for the prevention or treatment of a RSV infectionand/or amelioration of one or more symptoms of a RSV infection include,but are not limited to, about or 0.01 mg/kg to about or 300 mg/kg, suchas for example, about or 0.01 mg/kg, about or 0.1 mg/kg, about or 0.5mg/kg, about or 1 mg/kg, about or 5 mg/kg, about or 10 mg/kg, about or15 mg/kg, about or 20 mg/kg, about or 25 mg/kg, about or 30 mg/kg, aboutor 35 mg/kg, about or 40 mg/kg, about or 45 mg/kg, about or 50 mg/kg,about or 100 mg/kg, about or 150 mg/kg, about or 200 mg/kg, about or 250mg/kg, or about or 300 mg/kg.

In some examples, the anti-RSV antibodies or antigen-binding fragmentsthereof provided herein are administered to a subject at a dosageeffective to achieve a desired serum titer. In particular examples, theanti-RSV antibodies or antigen-binding fragments thereof provided hereinare administered for the prevention or treatment of a RSV infectionand/or amelioration of one or more symptoms of a RSV infection at anamount effective to achieve a serum titer of at least or about 1 μg/ml,at least or about 2 μg/ml, at least or about 3 μg/ml, at least or about4 μg/ml, at least or about 5 μg/ml, at least or about 6 μg/ml, at leastor about 7 μg/ml, at least or about 8 μg/ml, at least or about 9 μg/ml,at least or about 10 μg/ml, at least or about 15 μg/ml, at least orabout 20 μg/ml, at least or about 25 μg/ml, at least or about 30 μg/ml,at least or about 40 μg/ml, at least or about 50 μg/ml, at least orabout 60 μg/ml, at least or about 70 μg/ml, at least or about 80 μg/ml,at least or about 90 μg/ml, at least or about 100 μg/ml, at or about 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 15 days, 20 days, 25 days, 30 days, 35 days or 40 days followingadministration of a first dose of the antibody or antigen-bindingfragment thereof and prior to a subsequent dose of the antibody orantigen-binding fragment thereof.

In some examples, the anti-RSV antibodies or antigen-binding fragmentsthereof provided herein are administered by pulmonary delivery to asubject at a dosage effective to achieve a desired titer in anintubation sample, sputum or lavage from the lungs. In particularexamples, the anti-RSV antibodies or antigen-binding fragments thereofprovided herein are administered for the prevention or treatment of aRSV infection and/or amelioration of one or more symptoms of a RSVinfection at an amount effective to achieve a titer of 10 ng/mg (nganti-RSV antibody or antigen-binding fragment thereof per mg lungprotein) or about 10 ng/mg, 15 ng/mg or about 15 ng/mg, 20 ng/mg orabout 20 ng/mg, 25 ng/mg or about 25 ng/mg, 30 ng/mg or about 30 ng/mg,40 ng/mg or about 40 ng/mg, 50 ng/mg or about 50 ng/mg, 60 ng/mg orabout 60 ng/mg, 70 ng/mg or about 70 ng/mg, 80 ng/mg or about 80 ng/mg,90 ng/mg or about 90 ng/mg, 100 ng/mg or about 100 ng/mg, 110 ng/mg orabout 110 ng/mg, 120 ng/mg or about 120 ng/mg, 130 ng/mg or about 130ng/mg, 140 ng/mg or about 140 ng/mg, or 150 ng/mg or about 150 ng/mg inan intubation sample or lavage from the lungs at or about 10 days, 15days, 20 days, 25 days, 30 days, 35 days or 40 days followingadministration of a first dose of the antibody or antigen-bindingfragment thereof and prior to a subsequent dose of the antibody orantigen-binding fragment thereof.

For treatment of a viral infection, the dosage of the anti-RSVantibodies or antigen-binding fragments thereof can vary depending onthe type and severity of the disease. The anti-RSV antibodies orantigen-binding fragments thereof can be administered single dose, inmultiple separate administrations, or by continuous infusion. Forrepeated administrations over several days or longer, depending on thecondition, the treatment can be repeated until a desired suppression ofdisease symptoms occurs or the desired improvement in the patient'scondition is achieved. Repeated administrations can include increased ordecreased amounts of the anti-RSV antibody or antigen-binding fragmentthereof depending on the progress of the treatment. Other dosageregimens also are contemplated.

In some examples, the anti-RSV antibodies or antigen-binding fragmentsthereof provided herein are administered one time, two times, threetimes, four times, five times, six time, seven times, eight times, ninetimes, ten times or more per day or over several days. In particularexamples, the anti-RSV antibodies or antigen-binding fragments thereofprovided herein are administered one time, two times, three times, fourtimes, five times, six time, seven times, eight times, nine times, tentimes or more for the prevention or treatment of a RSV infection and/oramelioration of one or more symptoms of a RSV infection at an amounteffective to achieve a serum titer of at least or about 1 μg/ml, atleast or about 2 μg/ml, at least or about 3 μg/ml, at least or about 4μg/ml, at least or about 5 μg/ml, at least or about 6 μg/ml, at least orabout 7 μg/ml, at least or about 8 μg/ml, at least or about 9 μg/ml, atleast or about 10 μg/ml, at least or about 15 μg/ml, at least or about20 μg/ml, at least or about 25 μg/ml, at least or about 30 μg/ml, atleast or about 40 μg/ml, at least or about 50 μg/ml, at least or about60 μg/ml, at least or about 70 μg/ml, at least or about 80 μg/ml, atleast or about 90 μg/ml, at least or about 100 μg/ml, at or about 1 day,2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,15 days, 20 days, 25 days, 30 days, 35 days or 40 days followingadministration of a first dose, second dose, third dose, fourth dose,fifth dose, sixth dose, seventh dose, eighth dose, ninth dose, tenthdose of the antibody or antigen-binding fragment thereof and prior to asubsequent dose of the antibody or antigen-binding fragment thereof. Ina particular example, the anti-RSV antibodies or antigen-bindingfragments thereof provided herein are administered four times for theprevention or treatment of a RSV infection and/or amelioration of one ormore symptoms of a RSV infection at an amount effective to achieve aserum titer of at least or about 72 μg/ml at or about 1 day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 15 days,20 days, 25 days, 30 days, 35 days or 40 days following administrationof the fourth dose of the antibody or antigen-binding fragment thereofand prior to a subsequent dose of the antibody or antigen-bindingfragment thereof.

In some examples, the anti-RSV antibodies or antigen-binding fragmentsthereof are administered in a sequence of two or more administrations,where the administrations are separated by a selected time period. Insome examples, the selected time period is at least or about 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month,2 months, or 3 months.

In some examples, a prophylactically effective amount of an anti-RSVantibody or antigen-binding fragment thereof provided herein isadministered one or more times just prior to RSV season. In someexamples, a prophylactically effective amount of an anti-RSV antibody orantigen-binding fragment thereof provided herein is administered one ormore times just prior to RSV season and/or one or more times during RSVseason.

Therapeutic efficacy of a particular dosage or dosage regimen also canbe assessed, for example, by measurement of viral titer in the subjectprior to and following administration of one or more doses of theanti-RSV antibody or antigen-binding fragment thereof. Dosage amountsand/or frequency of administration can be modified depending on thedesired rate of clearance of the virus in the subject.

As will be understood by one of skill in the art, the optimal treatmentregimen will vary and it is within the scope of the treatment methods toevaluate the status of the disease under treatment and the generalhealth of the patient prior to, and following one or more cycles oftherapy in order to determine the optimal therapeutic dosage andfrequency of administration. It is to be further understood that for anyparticular subject, specific dosage regimens can be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thepharmaceutical formulations, and that the dosages set forth herein areexemplary only and are not intended to limit the scope thereof. Theamount of an anti-RSV antibody or antigen-binding fragment thereof to beadministered for the treatment of a disease or condition, for example aviral infection (e.g. a RSV virus infection), can be determined bystandard clinical techniques (e.g. viral titer or antigen detectionassays). In addition, in vitro assays and animal models can be employedto help identify optimal dosage ranges. Such assays can provide dosagesranges that can be extrapolated to administration to subjects, such ashumans. Methods of identifying optimal dosage ranges based on animalmodels are well known by those of skill in the art.

3. Routes of Administration

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be administered to a subject by any method known in the artfor the administration of polypeptides, including for example systemicor local administration. The anti-RSV antibodies or antigen-bindingfragments thereof can be administered by routes, such as parenteral(e.g., intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, or intracavity), topical, epidural, or mucosal (e.g.intranasal or oral). The anti-RSV antibodies or antigen-bindingfragments thereof can be administered externally to a subject, at thesite of the disease for exertion of local or transdermal action.Compositions containing anti-RSV antibodies or antigen-binding fragmentsthereof can be administered by any convenient route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa).Compositions containing anti-RSV antibodies or antigen-binding fragmentscan be administered together with other biologically active agents. Themode of administration can include topical or other administration of acomposition on, in or around areas of the body that may come on contactwith fluid, cells, or tissues that are infected, contaminated or haveassociated therewith a virus, such as a RSV virus. The anti-RSVantibodies or antigen-binding fragments thereof provided herein can beadministered by topical or aerosol routes for delivery directly totarget organs, such as the lungs (e.g. by pulmonary aerosol). In someexamples, the provided anti-RSV antibodies or antigen-binding fragmentsthereof can be administered as a controlled release formulation as suchas by a pump (see, e.g., Langer (1990) Science 249:1527-1533; Sefton(1987) CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al. (1980) Surgery88:507; and Saudek et al. (1989) N. Engl. J. Med. 321:574) or via theuse of various polymers known in the art and described elsewhere herein.In some examples, a controlled or sustained release system can be placedin proximity of the therapeutic target, for examples, the lungs, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

In particular examples, the provided anti-RSV antibodies orantigen-binding fragments thereof are administered by pulmonary delivery(see, e.g., U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos.WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903).Exemplary methods of pulmonary delivery are known in the art andinclude, but are not limited to, aerosol methods, such as inhalers(e.g., pressurized metered dose inhalers (MDI), dry powder inhalers(DPI), nebulizers (e.g., jet or ultrasonic nebulizers) and other singlebreath liquid systems), intratracheal instillation and insufflation. Insome examples, pulmonary delivery can be enhanced by co-administrationof or administration of a co-formulation containing the anti-RSVantibodies or antigen-binding fragments thereof provided herein and apermeation enhancer, such as, for example, surfactants, fatty acids,saccharides, chelating agents and enzyme inhibitors, such as proteaseinhibitors.

Appropriate methods for delivery, such as pulmonary delivery, can beselected by one of skill in the art based on the properties of thedosage amount of the anti-RSV antibody or antigen-binding fragmentthereof or the pharmaceutical composition containing the antibody orantigen-binding fragment thereof. Such properties include, but are notlimited to, solubility, hygroscopicity, crystallization properties,melting point, density, viscosity, flow, stability and degradationprofile.

In some examples, the anti-RSV antibodies or antigen-binding fragmentsthereof provided herein increase the efficacy mucosal immunizationagainst a virus. Thus, in particular examples the anti-RSV antibodies orantigen-binding fragments thereof are administered to a mucosal surface.For example, the anti-RSV antibodies or antigen-binding fragmentsthereof can be delivered via routes such as oral (e.g., buccal,sublingual), ocular (e.g., corneal, conjunctival, intravitreally,intra-aqueous injection), intranasal, genital (e.g., vaginal), rectal,pulmonary, stomachic, or intestinal. The anti-RSV antibodies orantigen-binding fragments thereof provided herein can be administeredsystemically, such as parenterally, for example, by injection or bygradual infusion over time or enterally (i.e., digestive tract). Theanti-RSV antibodies or antigen-binding fragments thereof provided hereinalso can be administered topically, such as for example, by topicalinstallation or application (e.g., intratracheal instillation andinsufflation using a bronchoscope or other artificial airway) of liquidsolutions, gels, ointments, powders or by inhalation (e.g., nasalsprays, inhalers (e.g., pressurized metered dose inhalers (MDI), drypowder inhalers (DPI), nebulizers (e.g., jet or ultrasonic nebulizers)and other single breath liquid systems)). Administration can be effectedprior to exposure to the virus or subsequent to exposure to the virus.

4. Combination Therapies

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be administered alone or in combination with one or moretherapeutic agents or therapies for the prophylaxis and/or treatment ofa disease or condition. For example, the provided anti-RSV antibodies orantigen-binding fragments thereof can be administered in combinationwith one or more antiviral agents for prophylaxis. In another example,the provided anti-RSV antibodies or antigen-binding fragments thereofcan be administered in combination with one or more antiviral agentstreatment of a viral infection, such as a respiratory viral infection.In some examples, the respiratory viral infection is a RSV infection.The antiviral agents can include agents to decrease and/or eliminate thepathogenic infection or agents to alleviate one or more symptoms of apathogenic infection. In some examples, a plurality of antibodies orantigen-binding fragments thereof (e.g., one or more antiviralantibodies) also can be administered in combination, where at least oneof the antibodies is an anti-RSV antibody or antigen-binding fragmentthereof provided herein. In some examples, a plurality of antibodies canbe administered in combination for the prophylaxis, where at least oneof the antibodies is an anti-RSV antibody or antigen-binding fragmentthereof provided herein. In other examples, a plurality of antibodiescan be administered in combination for treatment of a RSV infection ormultiple viral infections, where at least one of the antibodies is ananti-RSV antibody or antigen-binding fragment thereof provided herein.In some examples, the anti-RSV antibodies provided can be administeredin combination with one or more antiviral antibodies, which bind to andneutralize a virus, such as RSV. In some examples, the anti-RSVantibodies or antigen-binding fragments thereof provided can beadministered in combination with one or more antibodies, which caninhibit or alleviate one or more symptoms of a viral infection, such asa RSV infection. In some examples, two or more of the anti-RSVantibodies or antigen-binding fragments thereof provided herein areadministered in combination.

The one or more additional agents can be administered simultaneously,sequentially or intermittently with the anti-RSV antibody orantigen-binding fragment thereof. The agents can be co-administered withthe anti-RSV antibody or antigen-binding fragment thereof for example,as part of the same pharmaceutical composition or same method ofdelivery. In some examples, the agents can be co-administered with theanti-RSV antibody or antigen-binding fragment thereof at the same timeas the anti-RSV antibody or antigen-binding fragment thereof, but by adifferent means of delivery. The agents also can be administered at adifferent time than administration of the anti-RSV antibody orantigen-binding fragment thereof, but close enough in time to theadministration of the anti-RSV antibody or antigen-binding fragmentthereof to have a combined prophylactic or therapeutic effect. In someexamples, the one or more additional agents are administered subsequentto or prior to the administration of the anti-RSV antibody orantigen-binding fragment thereof separated by a selected time period. Insome examples, the time period is 1 day, 2 days, 3 days, 4 days, 5 days,6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months. Insome examples, the one ore more additional agents are administeredmultiple times and/or the anti-RSV antibody or antigen-binding fragmentthereof provided herein is administered multiple times.

In some examples, administration of the combination inhibits theincidence of RSV infection by at least or about 99%, at least or about95%, at least or about 90%, at least or about 85%, at least or about80%, at least or about 75%, at least or about 70%, at least or about65%, at least or about 60%, at least or about 55%, at least or about50%, at least or about 45%, at least or about 40%, at least or about35%, at least or about 30%, at least or about 25%, at least or about20%, at least or about 15%, or at least or about 10% relative to theincidence of RSV infection in the absence of the combination. In someexamples, administration of the combination decreases the severity ofone or more symptoms of RSV infection by at least or about 99%, at leastor about 95%, at least or about 90%, at least or about 85%, at least orabout 80%, at least or about 75%, at least or about 70%, at least orabout 65%, at least or about 60%, at least or about 55%, at least orabout 50%, at least or about 45%, at least or about 40%, at least orabout 35%, at least or about 30%, at least or about 25%, at least orabout 20%, at least or about 15%, or at least or about 10% relative tothe severity of the one or more symptoms of RSV infection in the absenceof the combination.

In some examples, the combination inhibits the binding of RSV to itshost cell receptor by at least or about 99%, at least or about 95%, atleast or about 90%, at least or about 85%, at least or about 80%, atleast or about 75%, at least or about 70%, at least or about 65%, atleast or about 60%, at least or about 55%, at least or about 50%, atleast or about 45%, at least or about 40%, at least or about 35%, atleast or about 30%, at least or about 25%, at least or about 20%, atleast or about 15%, or at least or about 10% relative to the binding ofRSV to its host cell receptor in the absence of the combination. In someexamples, the combination inhibits RSV replication by at least or about99%, at least or about 95%, at least or about 90%, at least or about85%, at least or about 80%, at least or about 75%, at least or about70%, at least or about 65%, at least or about 60%, at least or about55%, at least or about 50%, at least or about 45%, at least or about40%, at least or about 35%, at least or about 30%, at least or about25%, at least or about 20%, at least or about 15%, or at least or about10% relative to RSV replication in the absence of the combination.

Any therapy which is known to be useful, or which is or has been usedfor the prevention, management, treatment, or amelioration of a RSVinfection or one or more symptoms thereof can be used in combinationwith anti-RSV antibody or antigen-binding fragment thereof providedherein (see, e.g., Gilman et al., Goodman and Gilman's: ThePharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York,2001; The Merck Manual of Diagnosis and Therapy, Berkow, M. D. et al.(eds.), 17th Ed., Merck Sharp & Dohme Research Laboratories, Rahway,N.J., 1999; Cecil Textbook of Medicine, 20th Ed., Bennett and Plum(eds.), W. B. Saunders, Philadelphia, 1996, for information regardingtherapies (e.g., prophylactic or therapeutic agents) which have been orare used for preventing, treating, managing, or ameliorating a RSVinfection or one or more symptoms thereof). Examples of such agentsinclude, but are not limited to, immunomodulator) agents,anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g.,beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone,methylprednisolone, prednisolone, prednisone, hydrocortisone),glucocorticoids, steroids, non-steroidal anti-inflammatory drugs (e.g.aspirin, ibuprofen, diclofenac, and COX-2 inhibitors)), pain relievers,leukotriene antagonists (e.g., montelukast, methyl xanthines,zafirlukast, and zileuton), bronchodilators, such as β2-agonists (e.g.,bambuterol, bitolterol, clenbuterol, fenoterol, formoterol, indacaterol,isoetharine, metaproterenol, pirbuterol, procaterol, reproterol,rimiterol, salbutamol (Albuterol, Ventolin), levosalbutamol, salmeterol,tulobuterol and terbutaline) and anticholinergic agents (e.g.,ipratropium bromide and oxitropium bromide), sulphasalazine,penicillamine, dapsone, antihistamines, anti-malarial agents (e.g.,hydroxychloroquine), and antiviral agents. The anti-RSV antibodies orantigen-binding fragments thereof provided herein also can beadministered in combination with one or more therapies for the treatmentof a RSV infection, including but not limited to, administration ofintravenous infusion of immunoglobulin, administration of supplementaloxygen and fluids or assisted breathing. The anti-RSV antibodies orantigen-binding fragments thereof provided herein also can beadministered in combination with one or more agents that regulate lungmaturation and surfactant protein expression, such as, but not limitedto, glucocorticoids, PPARγ ligands, and vascular endothelial cell growthfactor (VEGF).

Exemplary antiviral agents that can be selected for combination therapywith an anti-RSV antibody or antigen-binding fragment thereof providedherein include, but are not limited to, antiviral compounds, antiviralproteins, antiviral peptides, antiviral protein conjugates and antiviralpeptide conjugates, including, but not limited to, nucleoside analogs,nucleotide analogs, immunomodulators (e.g. interferons) andimmunostimulants. Combination therapy using antibodies and/or anti-RSVantibodies and antigen-binding fragments provided herewith arecontemplated as is combination with the antibodies and/or anti-RSVantibodies and antigen-binding fragments provided herein with otheranti-RSV antibodies and anti-RSV antibodies and antigen-bindingfragments.

Exemplary antiviral agents for the treatment of virus infections thatcan be administered in combination with the anti-RSV antibodies orantigen-binding fragments thereof provided herein include, but are notlimited to, acyclovir, famciclovir, ganciclovir, penciclovir,valacyclovir, valganciclovir, idoxuridine, trifluridine, brivudine,cidofovir, docosanol, fomivirsen, foscarnet, tromantadine, imiquimod,podophyllotoxin, entecavir, lamivudine, telbivudine, clevudine,adefovir, tenofovir, boceprevir, telaprevir, pleconaril, arbidol,amantadine, rimantadine, oseltamivir, zanamivir, peramivir, inosine,interferon (e.g., Interferon alfa-2b, Peginterferon alfa-2a),ribavirin/taribavirin, abacavir, emtricitabine, lamivudine, didanosine,zidovudine, apricitabine, stampidine, elvucitabine, racivir, amdoxovir,stavudine, zalcitabine, tenofovir, efavirenz, nevirapine, etravirine,rilpivirine, loviride, delavirdine, atazanavir, fosamprenavir,lopinavir, darunavir, nelfinavir, ritonavir, saquinavir, tipranavir,amprenavir, indinavir, enfuvirtide, maraviroc, vicriviroc, PRO 140,ibalizumab, raltegravir, elvitegravir, bevirimat, vivecon, includingtautomeric forms, analogs, isomers, polymorphs, solvates, derivatives,or salts thereof.

Exemplary antiviral agents for the prophylaxis and/or treatment of RSVinfections that can be administered in combination with the anti-RSVantibodies or antigen-binding fragments thereof provided herein include,but are not limited to, ribavirin, NIH-351 (Gemini Technologies),recombinant RSV vaccine (Aviron), RSVf-2 (Intracel), F-50042 (PierreFabre), T-786 (Trimeris). VP-36676 (ViroPharma), RFI-641 (American HomeProducts). VP-14637 (ViroPharma). PEP-1 and PFP-2 (American HomeProducts). RSV vaccine (Avant Immunotherapeutics), F-50077 (PierreFabre), and other anti-RSV antibodies or antigen-binding fragmentsthereof.

The anti-RSV antibodies or antigen-binding fragments thereof providedherein also can be administered in combination with one or more agentscapable of stimulating cellular immunity, such as cellular mucosalimmunity. Any agent capable of stimulatory cellular immunity can beused. Exemplary immunostimulatory agents include, cytokines, such as,but not limited to, interferons (e.g., IFN-α, β, γ, ω), lymphokines andhematopoietic growth factors, such as, for example, GM-CSF (granulocytemacrophage colony stimulating factor), Interleukin-2 (IL-2),Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-7 (IL-7),Interleukin-10 (IL-10), Interleukin-12 (IL-12), Interleukin-14 (IL-14),and Tumor Necrosis Factor (TNF).

For combination therapies with anti-pathogenic agents, dosages for theadministration of such compounds are known in the art or can bedetermined by one skilled in the art according to known clinical factors(e.g., subject's species, size, body surface area, age, sex,immunocompetence, and general health, duration and route ofadministration, the kind and stage of the disease, and whether othertreatments, such as other anti-pathogenic agents, are being administeredconcurrently).

a. Antiviral Antibodies for Combination Therapy

The anti-RSV antibodies or antigen-binding fragments thereof providedherein can be administered in combination with one or more additionalantibodies or antigen-binding fragments thereof. In some examples, theone or more additional antibodies are antiviral antibodies. In someexamples, the one or more additional antibodies bind to a viral antigen.In some examples, the one or more additional antibodies bind to a viralantigen that is a surface protein, such as a viral capsid protein or aviral envelope protein. In some examples, the one or more additionalantibodies bind to a viral antigen that is expressed on the surface ofan infected cell. In some examples, the one or more additionalantibodies bind to a viral antigen that is expressed intracellularly(i.e., within an infected cell). In some examples, the one or moreadditional antibodies binds to a virus that causes respiratory disease,such as, but not limited to, RSV, parainfluenza virus (PIV) or humanmetapneumovirus (hMPV). Compositions containing the mixtures ofantibodies also are provided herein.

Antibodies for use in combination with an anti-RSV antibody orantigen-binding fragment thereof provided herein include, but are notlimited to, monoclonal antibodies, multispecific antibodies, syntheticantibodies, human antibodies, humanized antibodies, chimeric antibodies,intrabodies, single-chain Fvs (scFv), single chain antibodies, Fabfragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies provided herein), and epitope-binding fragments of any ofthe above. The antibodies for use in combination with an anti-RSVantibody or antigen-binding fragment thereof provided herein can be ofany type (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁,IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂) or subclass of immunoglobulin molecule.

Antibodies for use in combination with an anti-RSV antibody orantigen-binding fragment thereof provided herein can be from any animalorigin, including birds and mammals (e.g., human, murine, donkey, sheep,rabbit, goat, guinea pig, camel, horse, or chicken). Typically, theantibodies for use in combination with an anti-RSV antibody orantigen-binding fragment thereof provided herein are human or humanizedantibodies. The antibodies for use in combination with an anti-RSVantibody or antigen-binding fragment thereof provided herein can bemonospecific, bispecific, trispecific or of greater multispecificity.

The antibodies for use in combination with an anti-RSV antibody orantigen-binding fragment thereof provided herein can include derivativeantibodies that are modified, for example, by the attachment of any typeof molecule to the antibody or antigen-binding fragment thereof such asby covalent attachment. Exemplary antibody or antigen-binding fragmentthereof derivatives include antibodies that have been modified, forexample, by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein, orcontain heterologous Fc domain with higher affinities for the FcRNreceptor (see, e.g. U.S. Pat. No. 7,083,784). Any of numerous chemicalmodifications can be carried out by known techniques, including, but notlimited to, specific chemical cleavage, acetylation, formylation, orsynthesis in the presence of tunicamycin. Additionally, the derivativecan contain one or more non-classical amino acids.

The one or more additional antibodies for use in combination with ananti-RSV antibody or antigen-binding fragment thereof provided hereincan be administered simultaneously, sequentially or intermittently withthe anti-RSV antibody or antigen-binding fragment thereof. The one ormore additional antibodies can be co-administered with the anti-RSVantibody or antigen-binding fragment thereof, for example, as part ofthe same pharmaceutical composition or same method of delivery. In someexamples, the one or more additional antibodies can be co-administeredwith the anti-RSV antibody or antigen-binding fragment thereof at thesame time as the anti-RSV antibody or antigen-binding fragment thereof,but by a different means of delivery. The one or more additionalantibodies also can be administered at a different time thanadministration of the anti-RSV antibody or antigen-binding fragmentthereof provided herein, but close enough in time to the administrationof the anti-RSV antibody or antigen-binding fragment thereof to have acombined prophylactic or therapeutic effect. In some examples, the oneor more additional antibodies are administered subsequent to or prior tothe administration of the anti-RSV antibody or antigen-binding fragmentthereof separated by a selected time period. In some examples, the timeperiod is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2weeks, 3 weeks, 1 month, 2 months, or 3 months. In some examples, theone ore more additional antibodies are administered multiple timesand/or the anti-RSV antibody or antigen-binding fragment thereofprovided herein is administered multiple times.

i. Anti-RSV Antibodies

In some examples, the one or more additional antiviral antibodies areanti-RSV antibodies or antigen-binding fragments thereof. In someexamples, an anti-RSV antibody or antigen-binding fragment thereofprovided herein is administered in combination with the one or moreadditional anti-RSV antibodies or antigen-binding fragments thereof forthe prophylaxis and/or treatment of a RSV infection. Exemplary anti-RSVantibodies or antigen-binding fragments thereof for combination therapywith an anti-RSV antibody or antigen-binding fragment thereof providedherein include anti-RSV antibodies or antigen-binding fragments thereofthat immunospecifically bind to and neutralize RSV. In some examples,the one or more additional anti-RSV antibodies or antigen-bindingfragments thereof includes an antibody or antigen-binding fragmentthereof that immunospecifically binds to RSV A subtype and/or RSV Bsubtype.

In some examples, the one or more additional antiviral antibodies forcombination therapy with an anti-RSV antibody or antigen-bindingfragment provided herein includes an anti-RSV antibody that binds to aRSV attachment protein (e.g. having an amino acid sequence set forth inSEQ ID NO:276), a RSV RNA polymerase beta subunit large structuralprotein) (L protein) (e.g. having an amino acid sequence set forth inSEQ ID NO:276), a RSV nucleocapsid protein (e.g. having an amino acidsequence set forth in SEQ ID NO:277), a RSV nucleoprotein (N) (e.g.having an amino acid sequence set forth in SEQ ID NO:278), a RSVphosphoprotein P (e.g. having an amino acid sequence set forth in SEQ IDNO:279), a RSV matrix protein (e.g. having an amino acid sequence setforth in SEQ ID NO:280), a RSV small hydrophobic (SH) protein (e.g.having an amino acid sequence set forth in SEQ ID NO:281), a RSVRNA-dependent polymerase, a RSV F protein (e.g. having an amino acidsequence set forth in SEQ ID NO:282), a RSV G protein (e.g. having anamino acid sequence set forth in SEQ ID NO:283), or an allelic variantof any of the above. In particular examples, the one or more additionalantiviral antibodies includes an anti-RSV antibody that binds to a RSV Fprotein. In particular examples, the one or more additional antiviralantibodies that bind to a RSV F protein bind to the A, B, C, I, II, IV,V, or VI antigenic sites of a RSV F glycoprotein (see, e.g., Lopez etal. (1998) J. Virol. 72:6922-6928).

In some examples, the one or more additional antiviral antibodies forcombination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein includes, but is not limited to,palivizumab (SYNAGIS®), motavizumab (NUMAX®), AFFF, P12f2, P12f4, P11d4,A1e9, A12a6, A13c4, A17d4, A4B4, A8c7, 1X-493L1, FR H3-3F4, M3H9, Y10H6,DG, AFFF(1), 6H8, L1-7E5, L2-15B10, A13a11, A1h5, A4B4(1),A4B4L1FR-S28R, A4B4-F52S, (see U.S. Pat. Nos. 5,824,307 and 6,818,216),rsv6, rsv11, rsv13, rsv19, rsv21, rsv22, rsv23 (see U.S. Pat. No.6,685,942), RF-1, RF-2 (see U.S. Pat. No. 5,811,524), or antigen-bindingfragments thereof. In some examples, the one or more additionalantiviral antibodies for combination therapy includes an antibody orantigen-binding fragment thereof containing a V_(H) chain and/or V_(L)chain having the amino acid sequence of a V_(H) chain and/or V_(L) chainof palivizumab (SYNAGIS®), motavizumab (NUMAX®), AFFF, P12f2, P12f4,P11d4, A1e9, A12a6, A13c4, A17d4, A4B4, A8c7, 1X-493L1, FR H3-3F4, M3H9,Y10H6, DG, AFFF(1), 6H8, L1-7E5, L2-15B10, A13a11, A1h5, A4B4(1),A4B4L1FR-S28R, A4B4-F52S, rsv6, rsv11, rsv13, rsv19, rsv21, rsv22,rsv23, RF-1, or RF-2. In some examples, the one or more additionalantiviral antibodies for combination therapy includes an antibody orantigen-binding fragment thereof containing one or more CDRs ofpalivizumab (SYNAGIS®), motavizumab (NUMAX®), AFFF, P12f2, P12f4, P11d4,A1e9, A12a6, A13c4, A17d4, A4B4, A8c7, 1X-493L1, FR H3-3F4, M3H9, Y10H6,DG, AFFF(1), 6H8, L1-7E5, L2-15B10, A13a11, A1h5, A4B4(1),A4B4L1FR-S28R, A4B4-F52S, rsv6, rsv11, rsv13, rsv19, rsv21, rsv22,rsv23, RF-1, or RF-2. In some examples, the one or more additionalantiviral antibodies for combination therapy includes an antibody orantigen-binding fragment thereof containing one or more CDRs of from ananti-RSV mouse monoclonal antibody such as, but not limited to, MAbs1153, 1142, 1200, 1214, 1237, 1129, 1121, 1107, 1112, 1269, 1269, 1243(Beeler et al. (1989) J. Virology 63(7):2841-2950), MAb151 (Mufson etal. (1987) J. Clin. Microbiol. 25:1635-1539), MAbs 43-1 and 13-1 (Fernieet al. (1982) Proc. Soc. Exp. Biol. Med 171:266-271), MAbs 1436C, 1302A,1308F, and 1331H (Anderson et al. (1984) J. Clin. Microbiol.19:934-936). Additional exemplary antibodies or antigen-bindingfragments thereof that can be used for combination therapy with ananti-RSV antibody or antigen-binding fragment provided herein include,but are not limited to, anti-RSV antibodies or antigen-binding fragmentsthereof described in, for example, U.S. Pat. Nos. 6,413,771, 5,840,298,5,811,524, 6,656,467, 6,537,809, 7,364,742, 7,070,786, 5,955,364,7,488,477, 6,818,216, 5,824,307, 7,364,737, 6,685,942, and 5,762,905 andU.S. Patent Pub. Nos. 2007-0082002, 2005-0175986, 2004-0234528,2006-0198840, 2009-0110684, 2006-0159695, 2006-0013824, 2005-0288491,2005-0019758, 2008-0226630, 2009-0137003, and 2009-0092609.

In some examples, the one or more additional antiviral antibodies forcombination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein includes an antibody or antigen-bindingfragment thereof containing a V_(H) chain having an amino acid sequenceset forth in any of SEQ ID NOS:103, 113, 122, 131, 137, 144, 149, 155,161, 167, 172, 176, 179, 182, 186, 190, 194, 198, 201, 205, 210, 215,222, 226, 233, 239, 246, 252, 257, 362, and 366. In some examples, theone or more additional antiviral antibodies for combination therapy withan anti-RSV antibody or antigen-binding fragment thereof provided hereinincludes an antibody or antigen-binding fragment thereof containing aV_(H) domain having an amino acid sequence set forth in any of SEQ IDNOS:104, 114, 123, 132, 138, 145, 150, 156, 162, 168, 173, 187, 206,227, 232, 234, 240, 247, 253, and 258. In some examples, the one or moreadditional antiviral antibodies for combination therapy with an anti-RSVantibody or antigen-binding fragment thereof provided herein includes anantibody or antigen-binding fragment thereof containing a V_(H) CDR1having an amino acid sequence set forth in any of SEQ ID NOS:105, 115,124, 363, and 367. In particular examples, the one or more additionalantiviral antibodies for combination therapy with an anti-RSV antibodyor antigen-binding fragment thereof provided herein includes an antibodyor antigen-binding fragment thereof containing a V_(H) CDR1 having theamino acid sequence TSGMSVG (SEQ ID NO:105), TAGMSVG (SEQ ID NO:115),AYAMS (SEQ ID NO:363), or GYTMH (SEQ ID NO:367). In some examples, theone or more additional antiviral antibodies for combination therapy withan anti-RSV antibody or antigen-binding fragment thereof provided hereinincludes an antibody or antigen-binding fragment thereof containing aV_(H) CDR2 having an amino acid sequence set forth in any of SEQ IDNOS:106, 125, 133, 157, 236, 259, 364, and 368. In a particular example,the one or more additional antiviral antibodies for combination therapywith an anti-RSV antibody or antigen-binding fragment thereof providedherein includes an antibody or antigen-binding fragment thereofcontaining a V_(H) CDR2 having the amino acid sequence DIWWDDKKDYNPSLKS(SEQ ID NO:106) or DIWWDDKKHYNPSLKD (SEQ ID NO:125), GISGSGDSTDYADSVKG(SEQ ID NO:364), or SITGGSNFINYSDSVKG (SEQ ID NO:368). In some examples,the one or more additional antiviral antibodies for combination therapywith an anti-RSV antibody or antigen-binding fragment thereof providedherein includes an antibody or antigen-binding fragment thereofcontaining a V_(H) CDR3 having an amino acid sequence set forth in anyof SEQ ID NOS:107, 116, 126, 139, 188, 241, 365, and 369. In aparticular example, the one or more additional antiviral antibodies forcombination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein includes an antibody or antigen-bindingfragment thereof containing a V_(H) CDR3 having the amino acid sequenceSMITNWYFDV (SEQ ID NO:107), DMIFNFYFDV (SEQ ID NO:126),HLPDYWNLDYTRFFYYMDV (SEQ ID NO:365), or APIAPPYFDH (SEQ ID NO:369).

In some examples, the one or more additional antiviral antibodies forcombination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein includes an antibody or antigen-bindingfragment thereof containing a V_(L) chain having an amino acid sequenceset forth in any of SEQ ID NOS:108, 117, 127, 134, 140, 146, 152, 158,164, 169, 174, 177, 180, 183, 189, 191, 195, 199, 202, 207, 211, 216,220, 223, 228, 236, 242, 248, 254, 260, 263, 370, 374, and 378. In someexamples, the one or more additional antiviral antibodies forcombination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein includes an antibody or antigen-bindingfragment thereof containing a V_(L) domain having an amino acid sequenceset forth in any of SEQ ID NOS:109, 118, 128, 135, 141, 147, 153, 159,165, 170, 175, 178, 181, 184, 192, 196, 200, 203, 208, 212, 217, 221,224, 229, 237, 243, 249, 255, 261 and 264. In some examples, the one ormore additional antiviral antibodies for combination therapy with ananti-RSV antibody or antigen-binding fragment thereof provided hereinincludes an antibody or antigen-binding fragment thereof containing aV_(L) CDR1 having an amino acid sequence set forth in any of SEQ IDNOS:110, 119, 129, 142, 154, 166, 244, 250, 265, 371, 375, and 379. In aparticular example, the one or more additional antiviral antibodies forcombination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein includes an antibody or antigen-bindingfragment thereof containing a V_(L) CDR1 having the amino acid sequenceKCQLSVGYMH (SEQ ID NO:110), SASSRVGYMH (SEQ ID NO:154). RATQSISSNYLA(SEQ ID NO:371), KASQNINDNLA (SEQ ID NO:375), or RATQSVSNFLN (SEQ IDNO:379). In some examples, the one or more additional antiviralantibodies for combination therapy with an anti-RSV antibody orantigen-binding fragment thereof provided herein includes an antibody orantigen-binding fragment thereof containing a V_(L) CDR2 having an aminoacid sequence set forth in any of SEQ ID NOS:111, 120, 136, 143, 160,171, 185, 218, 225, 230, 238, 245, 251, 256, 262, 266, 372, 376, and380. In a particular example, the one or more additional antiviralantibodies for combination therapy with an anti-RSV antibody orantigen-binding fragment thereof provided herein includes an antibody orantigen-binding fragment thereof containing a V_(L) CDR2 having theamino acid sequence DTSKLAS (SEQ ID NO:111), DTLLLDS (SEQ ID NO:218),GASNRAT (SEQ ID NO:372), GASSRAT (SEQ ID NO:376), or DASTSQS (SEQ IDNO:380). In some examples, the one or more additional antiviralantibodies for combination therapy with an anti-RSV antibody orantigen-binding fragment thereof provided herein includes an antibody orantigen-binding fragment thereof containing a V_(L) CDR3 having an aminoacid sequence set forth in any of SEQ ID NOS:112, 121, 193, 373, 377,and 381. In a particular example, the one or more additional antiviralantibodies for combination therapy with an anti-RSV antibody orantigen-binding fragment thereof provided herein includes an antibody orantigen-binding fragment thereof containing a V_(L) CDR3 having theamino acid sequence FQGSGYPFT (SEQ ID NO:112), QQYDISPYT (SEQ IDNO:373), QQYGGSPYT (SEQ ID NO:377), or QASINTPL (SEQ ID NO:381).

In some examples, the anti-RSV antibody or antigen-binding fragmentthereof provided herein can be administered in combination withhyperimmune serum or immune globulin enriched for anti-RSV antibodies,such as, for example, RSV hyperimmune globulin (RSV IVIG; RESPIGAM®;MedImmune Inc, Gaithersburg, Md.; see, e.g., Groothius et al. (1993) NewEng. J. Med 329:1524-1530).

ii. Antibodies Against Other Respiratory Viruses

In some examples, the one or more additional antiviral antibodies forcombination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein includes an antibody or antigen-bindingfragment thereof to an respiratory virus other than RSV, for example,selected from among an anti-human metapneumovirus (hMPV) antibody, ananti-parainfluenzavirus (PIV) antibody, an anti-avian pneumovirus (APV)antibody or other antiviral antibody known in the art.

In some examples, where the one or more additional antiviral antibodiesfor combination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein is an anti-PIV antibody, an antibodythat immunospecifically binds to a PIV antigen, such as, for example, aPIV nucleocapsid phosphoprotein, a PIV fusion (F) protein, a PIVphosphoprotein, a PIV large (L) protein, a PIV matrix (M) protein, a PIVhemagglutinin-neuraminidase (HN) glycoprotein, a PIV RNA-dependent RNApolymerase, a PIV Y1 protein, a PIV D protein, a PIV C protein, or anallelic variant of any of the above. In particular examples, the PIVantigen is PIV F protein. In some examples, the anti-PIV antibody is anantibody that immunospecifically binds to an antigen of human PIV type1, human PIV type 2, human PIV type 3, and/or human PIV type 4.

In some examples, where the one or more additional antiviral antibodiesfor combination therapy with an anti-RSV antibody or antigen-bindingfragment thereof provided herein is an anti-hMPV antibody, an antibodythat immunospecifically binds to a hMPV antigen, such as, for example, ahMPV nucleoprotein, a hMPV phosphoprotein, a hMPV matrix protein, a hMPVsmall hydrophobic protein, a hMPV RNA-dependent RNA polymerase, a hMPV Fprotein, a hMPV G protein, or an allelic variant of any of the above. Inparticular examples, the hMPV antigen is PIV F protein. In someexamples, the anti-hMPV antibody is an antibody that immunospecificallybinds to an antigen of hMPV type A and/or hMPV type B. In some examples,the anti-hMPV antibody is an antibody that immunospecifically binds toan antigen of hMPV sub-type A1 and/or A2 and/or hMPV sub-type B1 and/orB2.

Antibodies administered in combination with an anti-RSV antibody orantigen-binding fragment thereof provided herein can be any type ofantibody or antigen-binding fragment known in the art. For example, anantibody or antigen-binding fragment thereof administered in combinationwith an anti-RSV antibody or antigen-binding fragment thereof providedherein can include, but is not limited to, a monoclonal antibody, ahuman antibody, a non-human antibody, a recombinantly produced antibody,a chimeric antibody, a humanized antibody, a multispecific antibody(e.g., a bispecific antibody), an intrabody, and an antibody fragment,such as, but not limited to, a Fab fragment, a Fab′ fragment, a F(ab′)₂fragment, a Fv fragment, a disulfide-linked Fv (dsFv), a Fd fragment, aFd′ fragment, a single-chain Fv (scFv), a single-chain Fab (scFab), adiabody, an anti-idiotypic (anti-Id) antibody, or antigen-bindingfragments of any of the above. Antibodies administered in combinationwith an anti-RSV antibody provided herein can include members of anyimmunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA and IgY), any class(e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass (e.g., IgG2aand IgG2b).

In some examples, administration of the combination of antiviralantibodies or antigen-binding fragments inhibits the incidence of RSVinfection by at least or about 99%, at least or about 95%, at least orabout 90%, at least or about 85%, at least or about 80%, at least orabout 75%, at least or about 70%, at least or about 65%, at least orabout 60%, at least or about 55%, at least or about 50%, at least orabout 45%, at least or about 40%, at least or about 35%, at least orabout 30%, at least or about 25%, at least or about 20%, at least orabout 15%, or at least or about 10% relative to the incidence of RSVinfection in the absence of the anti-RSV antibody or antigen-bindingfragment. In some examples, administration of the combination ofantiviral antibodies or antigen-binding fragments decreases the severityof one or more symptoms of RSV infection by at least or about 99%, atleast or about 95%, at least or about 90%, at least or about 85%, atleast or about 80%, at least or about 75%, at least or about 70%, atleast or about 65%, at least or about 60%, at least or about 55%, atleast or about 50%, at least or about 45%, at least or about 40%, atleast or about 35%, at least or about 30%, at least or about 25%, atleast or about 20%, at least or about 15%, or at least or about 10%relative to the severity of the one or more symptoms of RSV infection inthe absence of the combination of antiviral antibodies orantigen-binding fragments.

In some examples, the combination of antiviral antibodies orantigen-binding fragments inhibits the binding of RSV to its host cellreceptor by at least or about 99%, at least or about 95%, at least orabout 90%, at least or about 85%, at least or about 80%, at least orabout 75%, at least or about 70%, at least or about 65%, at least orabout 60%, at least or about 55%, at least or about 50%, at least orabout 45%, at least or about 40%, at least or about 35%, at least orabout 30%, at least or about 25%, at least or about 20%, at least orabout 15%, or at least or about 10% relative to the binding of RSV toits host cell receptor in the absence of the combination of antiviralantibodies or antigen-binding fragments. In some examples, thecombination of antiviral antibodies or antigen-binding fragmentsinhibits RSV replication by at least or about 99%, at least or about95%, at least or about 90%, at least or about 85%, at least or about80%, at least or about 75%, at least or about 70%, at least or about65%, at least or about 60%, at least or about 55%, at least or about50%, at least or about 45%, at least or about 40%, at least or about35%, at least or about 30%, at least or about 25%, at least or about20%, at least or about 15%, or at least or about 10% relative to RSVreplication in the absence of the combination of antiviral antibodies orantigen-binding fragments.

5. Gene Therapy

In some examples, nucleic acids comprising sequences encoding theanti-RSV antibodies, antigen-binding fragments and/or derivativesthereof, are administered to treat, prevent or ameliorate one or moresymptoms associated with RSV infection, by way of gene therapy. Genetherapy refers to therapy performed by the administration to a subjectof an expressed or expressible nucleic acid. In this example, thenucleic acids produce their encoded antibody or antigen-binding fragmentthereof that mediates a prophylactic or therapeutic effect.

Any of the methods for gene therapy available in the art can be employedfor administration of nucleic acid encoding the anti-RSV antibodies,antigen-binding fragments and/or derivatives thereof. Exemplary methodsare described below.

For general reviews of the methods of gene therapy, see, for example,Goldspiel et al. (1993) Clinical Pharmacy 12:488-505; Wu and Wu (1991)Biotherapy 3:87-95; Tolstoshev (1993) Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan (1993) Science 260:926-932; Morgan and Anderson(1993) Ann. Rev. Biochem. 62:191-217; and TIBTECH 11(5):155-215. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In some examples, a composition provided herein contains nucleic acidsencoding an anti-RSV antibody, an antigen-binding fragment and/orderivative thereof, where the nucleic acids are part of an expressionvector that expresses the anti-RSV antibody, antigen-binding fragmentand/or derivative thereof in a suitable host. In particular, suchnucleic acids have promoters, such as heterologous promoters, operablylinked to the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies (1989) Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al. (1989) Nature 342:435-438). In someexamples, the expressed antibody molecule is a single chain antibody. Insome examples, the nucleic acid sequences include sequences encoding theheavy and light chains, or fragments thereof, of the antibody. In aparticular example, the nucleic acid sequences include sequencesencoding an anti-RSV Fab fragment. In a particular example, the nucleicacid sequences include sequences encoding a full-length anti-RSVantibody. In some examples, the encoded anti-RSV antibody is a chimericantibody.

Delivery of the nucleic acids into a subject can be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In some examples, the nucleic acid sequences are directly administeredin vivo, where it is expressed to produce the encoded product. This canbe accomplished by any of numerous methods known in the art, forexample, by constructing them as part of an appropriate nucleic acidexpression vector and administering it so that they becomeintracellular, for example, by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun: Biolistic. Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432)which can be used, for example, to target cell types specificallyexpressing the receptors. In some examples, nucleic acid-ligandcomplexes can be formed in which the ligand contains a fusogenic viralpeptide to disrupt endosomes, allowing the nucleic acid to avoidlysosomal degradation. In some examples, the nucleic acid can betargeted in vivo for cell specific uptake and expression, by targeting aspecific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/203 16; WO93/14188, and WO 93/20221). Alternatively, the nucleicacid can be introduced intracellularly and incorporated within host cellDNA for expression, by homologous recombination (Koller and Smithies(1989) Proc. Natl. Acad. Sci. USA 86:8932-8935; and Zijlstra et al.(1989) Nature 342:435-438).

In some examples, viral vectors that contain nucleic acid sequencesencoding an anti-RSV antibody, antigen-binding fragments and/orderivatives thereof are used. For example, a retroviral vector can beused (see, e.g., Miller et al. (1993) Meth. Enzymol. 217:581-599).Retroviral vectors contain the components necessary for the correctpackaging of the viral genome and integration into the host cell DNA.The nucleic acid sequences encoding the antibody or antigen-bindingfragment thereof to be used in gene therapy are cloned into one or morevectors, which facilitate delivery of the gene into a subject. Moredetail about retroviral vectors can be found, for example, in Boesen etal. (1994) Biotherapy 6:291-302. Other references illustrating the useof retroviral vectors in gene therapy include, for example, Clowes etal. (1994) J. Clin. Invest. 93:644-651; Klein et al. (1994) Blood83:1467-1473; Salmons and Gunzberg (1993) Human Gene Therapy 4:129-141;and Grossman and Wilson (1993) Curr. Opin, in Genetics and Devel.3:110-114.

Adenoviruses also are viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems include the liver, central nervoussystem, endothelial cells, and muscle. Adenoviruses have the advantageof being capable of infecting non-dividing cells. Kozarsky and Wilson(1993) Current Opinion in Genetics and Development 3:499-503 present areview of adenovirus-based gene therapy. Bout et al. (1994) Human GeneTherapy 5:3-10 demonstrated the use of adenovirus vectors to transfergenes to the respiratory epithelia of rhesus monkeys. Other instances ofthe use of adenoviruses in gene therapy can be found, for examples, inRosenfeld et al. (1991) Science 252:431-434; Rosenfeld et al. (1992)Cell 68:143-155; Mastrangeli et al. (1993) J. Clin. Invest. 91:225-234;PCT Publication WO94/12649; and Wang et al. (1995) Gene Therapy2:775-783. In a particular example, adenovirus vectors are used todeliver nucleic acid encoding the anti-RSV antibodies, antigen-bindingfragments and/or derivatives thereof provided herein.

Adeno-associated virus (AAV) also can be used in gene therapy (Walsh etal. (1993) Proc. Soc. Exp. Biol. Med. 204:289-300; and U.S. Pat. No.5,436,146). In a particular example, adeno-associated virus (AAV)vectors are used to deliver nucleic acid encoding the anti-RSVantibodies, antigen-binding fragments and/or derivatives thereofprovided herein.

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Generally,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thecells expressing the gene are then delivered to a subject.

In some examples, the nucleic acid encoding an anti-RSV antibody,antigen-binding fragments and/or derivatives thereof provided herein isintroduced into a cell prior to administration in vivo of the resultingrecombinant cell. Such introduction can be carried out by any methodknown in the art, including, but not limited to, transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer, andspheroplast fusion. Numerous techniques are known in the art for theintroduction of foreign genes into cells (see, e.g., Loeffler and Behr(1993) Meth. Enzymol. 217:599-618: Cohen et al. (1993) Meth. Enzymol.217:618-644; Cline (1985) Pharmacol. Ther. 29:69-92) and can be used forthe administration of nucleic acid encoding an anti-RSV antibody,antigen-binding fragment and/or derivative thereof provided herein,provided that the necessary developmental and physiological functions ofthe recipient cells are not disrupted. The technique provides for thestable transfer of the nucleic acid to the cell, so that the nucleicacid is expressible by the cell and typically heritable and expressibleby its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) can be administered intravenously. The amountof cells for administration depends on various factors, including, forexample, the desired prophylactic and/or therapeutic effect and patientstate, and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include, but arenot limited to, epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, forexample, as obtained from bone marrow, umbilical cord blood, peripheralblood, and fetal liver. In particular examples, the cell used for genetherapy is autologous to the subject.

In some examples in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an anti-RSV antibody, antigen-bindingfragment and/or derivative thereof provided herein are introduced intothe cells such that they are expressible by the cells or their progeny,and the recombinant cells are then administered in vivo for therapeuticeffect. In a particular example, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can be used (see e.g., PCT Publication WO 94/08598; Stemple andAnderson (1992) Cell 7 1:973-985; Rheinwald (1980) Meth. Cell Bio.21A:229; and Pittelkow and Scott (1986) Mayo Clinic Proc. 61:771).

In a particular example, the nucleic acid to be introduced for purposesof gene therapy contains an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

J. PHARMACEUTICAL COMPOSITIONS, COMBINATIONS AND ARTICLES OFMANUFACTURE/KITS

1. Pharmaceutical Compositions

Provided herein are pharmaceutical compositions containing an anti-RSVantibody or antigen-binding fragment thereof provided herein. Thepharmaceutical composition can be used for therapeutic, prophylactic,and/or diagnostic applications. The anti-RSV antibodies orantigen-binding fragments thereof provided herein can be formulated witha pharmaceutical acceptable carrier or diluent. Generally, suchpharmaceutical compositions utilize components which will notsignificantly impair the biological properties of the antibody orantigen-binding fragment thereof, such as the binding of to its specificepitope (e.g. binding to an epitope on a RSV F protein). Each componentis pharmaceutically and physiologically acceptable in the sense of beingcompatible with the other ingredients and not injurious to the patient.The formulations can conveniently be presented in unit dosage form andcan be prepared by methods well known in the art of pharmacy, includingbut not limited to, tablets, pills, powders, liquid solutions orsuspensions (e.g., including injectable, ingestible and topicalformulations (e.g., eye drops, gels or ointments), aerosols (e.g., nasalsprays), liposomes, suppositories, injectable and infusible solution andsustained release forms. See, e.g., Gilman, et al. (eds. 1990) Goodmanand Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed.,Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.(1990). Mack Publishing Co., Easton. Pa.; Avis, et al (eds. 1993)Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, NY;Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Tablets,Dekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Disperse Systems, Dekker, NY. When administered systematically,the therapeutic composition is sterile, pyrogen-free, generally free ofparticulate matter, and in a parenterally acceptable solution having dueregard for pH, isotonicity, and stability. These conditions are known tothose skilled in the art. Methods for preparing parenterallyadministrable compositions are well known or will be apparent to thoseskilled in the art and are described in more detail in, e.g.,“Remington: The Science and Practice of Pharmacy (Formerly Remington'sPharmaceutical Sciences),” 19th ed., Mack Publishing Company, Easton,Pa. (1995).

Pharmaceutical compositions provided herein can be in various forms,e.g., in solid, semi-solid, liquid, powder, aqueous, or lyophilizedform. Examples of suitable pharmaceutical carriers are known in the artand include but are not limited to water, buffering agents, salinesolutions, phosphate buffered saline solutions, various types of wettingagents, sterile solutions, alcohols, gum arabic, vegetable oils, benzylalcohols, gelatin, glycerin, carbohydrates such as lactose, sucrose,amylose or starch, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, powders,among others. Pharmaceutical compositions provided herein can containother additives including, for example, antioxidants, preservatives,antimicrobial agents, analgesic agents, binders, disintegrants,coloring, diluents, excipients, extenders, glidants, solubilizers,stabilizers, tonicity agents, vehicles, viscosity agents, flavoringagents, emulsions, such as oil/water emulsions, emulsifying andsuspending agents, such as acacia, agar, alginic acid, sodium alginate,bentonite, carbomer, carrageenan, carboxymethylcellulose, cellulose,cholesterol, gelatin, hydroxyethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose, methylcellulose, octoxynol 9, oleylalcohol, povidone, propylene glycol monostearate, sodium lauryl sulfate,sorbitan esters, stearyl alcohol, tragacanth, xanthan gum, andderivatives thereof, solvents, and miscellaneous ingredients such ascrystalline cellulose, microcrystalline cellulose, citric acid, dextrin,dextrose, liquid glucose, lactic acid, lactose, magnesium chloride,potassium metaphosphate, starch, among others (see, generally, AlfonsoR. Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20thEdition. Baltimore, Md.: Lippincott Williams & Wilkins). Such carriersand/or additives can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Stabilizing agents suchas lipids, nuclease inhibitors, polymers, and chelating agents canpreserve the compositions from degradation within the body.

Pharmaceutical compositions suitable for use include compositionswherein one or more anti-RSV antibodies are contained in an amounteffective to achieve their intended purpose. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art. Therapeutically effective dosages can be determinedby using in vitro and in vivo methods as described herein. Accordingly,an anti-RSV antibody or antigen-binding fragment thereof providedherein, when in a pharmaceutical preparation, can be present in unitdose forms for administration.

An anti-RSV antibody or antigen-binding fragment thereof provided hereincan be lyophilized for storage and reconstituted in a suitable carrierprior to use. This technique has been shown to be effective withconventional immunoglobulins and protein preparations and art-knownlyophilization and reconstitution techniques can be employed.

An anti-RSV antibody or antigen-binding fragment thereof provided hereincan be provided as a controlled release or sustained releasecomposition. Polymeric materials are known in the art for theformulation of pills and capsules which can achieve controlled orsustained release of the antibodies or antigen-binding fragments thereofprovided herein (see, e.g., Medical Applications of Controlled Release,Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); ControlledDrug Bioavailability, Drug Product Design and Performance, Smolen andBall (eds.), Wiley, New York (1984); Ranger and Peppas (1983) J.,Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al. (1985)Science 228:190; During et al. (1989) Ann. Neurol. 25:351; Howard et al.(1989) J. Neurosurg. 7 1:105; U.S. Pat. Nos. 5,679,377, 5,916,597,5,912,015, 5,989,463, 5,128,326; PCT Publication Nos. WO 99/15154 and WO99/20253). Examples of polymers used in sustained release formulationsinclude, but are not limited to, poly(2-hydroxy ethyl methacrylate),poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinylacetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides)(PLGA), and polyorthoesters. Generally, the polymer used in a sustainedrelease formulation is inert, free of leachable impurities, stable onstorage, sterile, and biodegradable. Any technique known in the art forthe production of sustained release formulation can be used to produce asustained release formulation containing one or more anti-RSV antibodiesor antigen-binding fragments provided herein.

In some examples, the pharmaceutical composition contains an anti-RSVantibody or antigen-binding fragment thereof provided herein and one ormore additional antibodies. In some examples, the one or more additionalantibodies includes, but is not limited to, palivizumab (SYNAGIS®), andderivatives thereof, such as, but not limited to, motavizumab (NUMAX®),AFFF, P12f2, P12f4, P11d4, A1e9, A12a6, A13c4, A17d4, A4B4, A8c7,1X-493L1, FR H3-3F4, M3H9, Y10H6, DG, AFFF(1), 6H8, L1-7E5, L2-15B10,A13a11, A1h5, A4B4(1), A4B4L1FR-S28R, and A4B4-F52S (see U.S. Pat. Nos.5,824,307 and 6,818,216), rsv6, rsv11, rsv13, rsv19, rsv21, rsv22, rsv23(see, e.g. U.S. Pat. Nos. 5,824,307, 6,685,942 and 6,818,216), a humananti-RSV antibody, such as, but not limited to, rsv6, rsv11, rsv13,rsv19 (i.e. Fab 19), rsv21, rsv22, rsv23, RF-1, RF-2 (see, e.g. U.S.Pat. Nos. 6,685,942 and 5,811,524), a humanized antibody derived from ananti-RSV mouse monoclonal antibody such as, but not limited to, MAbs1153, 1142, 1200, 1214, 1237, 1129, 1121, 1107, 1112, 1269, 1269, 1243(Beeler et al. (1989) J. Virology 63(7):2841-2950), MAb151 (Mufson etal. (1987) J. Clin. Microbiol. 25:1635-1539), MAbs 43-1 and 13-1 (Fernieet al. (1982) Proc. Soc. Exp. Biol. Med. 171:266-271), MAbs 1436C,1302A, 1308F, and 1331H (Anderson et al. (1984) J. Clin. Microbiol.19:934-936), or antigen-binding fragments thereof. Additional exemplaryantibodies or antigen-binding fragments thereof that can be used in apharmaceutical composition containing an anti-RSV antibody orantigen-binding fragment thereof provided herein include, but are notlimited to, anti-RSV antibodies or antigen-binding fragments thereofdescribed in, for example, U.S. Pat. Nos. 6,413,771, 5,840,298,5,811,524, 6,656,467, 6,537,809, 7,364,742, 7,070,786, 5,955,364,7,488,477, 6,818,216, 5,824,307, 7,364,737, 6,685,942, and 5,762,905 andU.S. Patent Pub. Nos. 2007-0082002, 2005-0175986, 2004-0234528,2006-0198840, 2009-0110684, 2006-0159695, 2006-0013824, 2005-0288491,2005-0019758, 2008-0226630, 2009-0137003, and 2009-0092609.

2. Articles of Manufacture/Kits

Pharmaceutical compositions of anti-RSV antibodies or nucleic acidsencoding anti-RSV antibodies, or a derivative or a biologically activeportion thereof can be packaged as articles of manufacture containingpackaging material, a pharmaceutical composition which is effective forprophylaxis (i.e. vaccination, passive immunization) and/or treating theRSV-mediated disease or disorder, and a label that indicates that theantibody or nucleic acid molecule is to be used for vaccination and/ortreating the disease or disorder. The pharmaceutical compositions can bepackaged in unit dosage forms contain an amount of the pharmaceuticalcomposition for a single dose or multiple doses. The packagedcompositions can contain a lyophilized powder of the pharmaceuticalcompositions containing the anti-RSV antibodies or antigen-bindingfragments thereof provided, which can be reconstituted (e.g. with wateror saline) prior to administration.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. Examples of pharmaceuticalpackaging materials include, but are not limited to, blister packs,bottles, tubes, inhalers, inhalers (e.g., pressurized metered doseinhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet orultrasonic nebulizers) and other single breath liquid systems), pumps,bags, vials, containers, syringes, bottles, and any packaging materialsuitable for a selected formulation and intended mode of administrationand treatment. The pharmaceutical composition also can be incorporatedin, applied to or coated on a barrier or other protective device that isused for contraception from infection.

The anti-RSV antibodies or antigen-binding fragments thereof, nucleicacid molecules encoding the antibodies thereof, pharmaceuticalcompositions or combinations provided herein also can be provided askits. Kits can optionally include one or more components such asinstructions for use, devices and additional reagents (e.g., sterilizedwater or saline solutions for dilution of the compositions and/orreconstitution of lyophilized protein), and components, such as tubes,containers and syringes for practice of the methods. Exemplary kits caninclude the anti-RSV antibodies or antigen-binding fragments thereofprovided herein, and can optionally include instructions for use, adevice for administering the anti-RSV antibodies or antigen-bindingfragments thereof to a subject, a device for detecting the anti-RSVantibodies or antigen-binding fragments thereof in a subject, a devicefor detecting the anti-RSV antibodies or antigen-binding fragmentsthereof in samples obtained from a subject, and a device foradministering an additional therapeutic agent to a subject.

The kit can, optionally, include instructions. Instructions typicallyinclude a tangible expression describing the anti-RSV antibodies orantigen-binding fragments thereof and, optionally, other componentsincluded in the kit, and methods for administration, including methodsfor determining the proper state of the subject, the proper dosageamount, dosing regimens, and the proper administration method foradministering the anti-RSV antibodies or antigen-binding fragmentsthereof. Instructions also can include guidance for monitoring thesubject over the duration of the treatment time

Kits also can include a pharmaceutical composition described herein andan item for diagnosis. For example, such kits can include an item formeasuring the concentration, amount or activity of the selected anti-RSVantibody or antigen-binding fragment thereof in a subject.

In some examples, the anti-RSV antibody or antigen-binding fragmentthereof is provided in a diagnostic kit for the detection of RSV in anisolated biological sample (e.g., a fluid sample, such as blood, sputum,lavage, lung intubation sample, saliva, urine or lymph obtained from asubject). In some examples, the diagnostic kit contains a panel of oneor more anti-RSV antibodies or antigen-binding fragments thereof and/orone or more control antibodies (i.e. non-RSV binding antibodies), whereone or more antibodies in the panel is an anti-RSV antibody orantigen-binding fragment provided herein.

Kits provided herein also can include a device for administering theanti-RSV antibodies or antigen-binding fragments thereof to a subject.Any of a variety of devices known in the art for administeringmedications to a subject can be included in the kits provided herein.Exemplary devices include, but are not limited to, an inhaler (e.g.,pressurized metered dose inhaler (MDI), dry powder inhaler (DPI),nebulizer (e.g., jet or ultrasonic nebulizers) and other single breathliquid system), a hypodermic needle, an intravenous needle, a catheter,and a liquid dispenser such as an eyedropper. Typically the device foradministering the anti-RSV antibodies or antigen-binding fragmentsthereof of the kit will be compatible with the desired method ofadministration of the anti-RSV antibodies or antigen-binding fragmentsthereof. For example, an anti-RSV antibody or antigen-binding fragmentthereof to be delivered by pulmonary administration can be included in akit with or contained in an inhaler or a nebulizer.

3. Combinations

Provided are combinations of the anti-RSV antibodies or antigen-bindingfragments thereof provided herein and a second agent, such as a secondanti-RSV antibody or antigen-binding fragment thereof or othertherapeutic or diagnostic agent. A combination can include any anti-RSVantibody or antigen-binding fragment thereof or reagent for effectingtherapy thereof in accord with the methods provided herein. For example,a combination can include any anti-RSV antibody or antigen-bindingfragment thereof and an antiviral agent. Combinations also can includean anti-RSV antibody or antigen-binding fragment thereof provided hereinwith one or more additional therapeutic antibodies. Combinations of theanti-RSV antibodies or antigen-binding fragments thereof provided alsocan contain pharmaceutical compositions containing the anti-RSVantibodies or antigen-binding fragments thereof or host cells containingnucleic acids encoding the anti-RSV antibodies or antigen-bindingfragments thereof as described herein. The combinations provided hereincan be formulated as a single composition or in separate compositions.

K. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Expression of RSV F Protein

In this example, the RSV fusion protein (F protein) from RespiratorySyncytial Virus strain A2 was expressed and purified by capture on ELISAplates using anti-RSV monoclonal antibody clone 2F7, which recognizesboth the F0 and F1 subunits of the fusion glycoprotein. In the firstexample, recombinant RSV F protein was cloned and expressed in 293Fcells. In the second example, native RSV F protein was expressed byinfection of HEp-2 cells with RSV A2 strain.

A. Recombinant RSV F Protein

In this example, the gene encoding the RSV F protein from the A2 RSVstrain was cloned and expressed. The RSV A2 F gene (SEQ ID NO:21),containing only the extracellular domain was synthesized according tostandard DNA synthesis protocols by GeneArt (Burlingame, Calif.). TheRSV A2 F gene was engineered to contain a Kozak sequence (nucleotides7-16 of SEQ ID NO:21), a c-myc sequence (nucleotides 1600-1629 of SEQ IDNO:21), and a 6×-His tag (nucleotides 1645-417 of SEQ ID NO:21).Additionally, NheI (SEQ ID NO:22) and HindIII (SEQ ID NO:23) restrictionsites were engineered at the 5′ and 3′ ends, respectively, to allowcloning into an expression vector. The DNA was digested using standardmolecular biology techniques and ligated into the similarly digestedmammalian expression vector pcDNA™3.1/myc-His(−) C (SEQ ID NO:24,Invitrogen). The vector containing the RSV A2 F gene was s transformedinto electrocompetent XL1-Blue cells (Strategene). Individual colonieswere selected and grown, and the plasmid DNA was purified. The presenceof the RSV A2 F gene insert in the isolated vector was verified by DNAsequencing, and one clone containing the insert was used to producelarge-scale preparations of DNA (Megaprep kit, Qiagen).

The RSV A2 F protein was expressed in mammalian cells using theFreeStyle™ 293 Expression System (Invitrogen) according to themanufacturer's instructions. Briefly, 3×10⁷ cells were co-transfectedwith 30 μg of RSV A2 F/pcDNA3.1/myc-His(−) C plasmid DNA and 5 μgpAdVAntage (Promega) and incubated at 37° C. for 72 hrs. Cells werepelleted by centrifugation and 3 mL of cold lysis buffer (300 mM NaCl,50 mM NaH₂PO₄, 1% TRITON® X-100, Complete™ Protease Inhibitor cocktail(Cat. No, sc-29131, Santa Cruz), pH 8) was added to for every 3×10⁷ RSVF-transfected 293-F cells. The mixture was rocked at 4° C. for 30 minfollowed by centrifugation at 14,000 rpm for 30 min at 4° C. The clearedsupernatant was transferred to a fresh tube and frozen at −80° C. untilready for use. Prior to capture on an ELISA plate, the supernatant wasthawed, briefly centrifuged and diluted 1:1 v/v with PBS containing 0.8%nonfat dry milk (final concentration of 0.4% nonfat dry milk).

B. Native RSV F Protein

In this example, native RSV F protein from the RSV A2 strain (SEQ IDNO:382) was purified from RSV infected HEp-2 cells as follows. Briefly,HEp-2 cells are seeded in a ten-layer cell culture stacker (Corning3270) using complete EMEM (ATCC 30-2003; containing 10% FBS, 1%L-glutamine, and 1% pen-Strep) and incubated at 37° C., and 5% CO₂. Oncethe cells reached 80% confluence, the HEp-2 monolayer was infected withthe RSV A2 virus (ATCC VR-1540) at an multiplicity of infection (MOI) of0.01-0.1. The infected cells were cultured for 3-5 days until apparentcell syncytia was observed. The infected cells were washed once with PBSand the cells were harvested by adding 500 mL PBS with 5 mM EDTA to theculture stacker and incubating at 37° C. for 1 hr. Cells were collectedinto 50 mL conical tubes (5×10⁷ cells per tube) and pelleted bycentrifugation. The cell pellets were washed 2× with PBS and centrifugedat 1200 rpm for 5 minutes. The cell pellets were stored at −20° C. untilfurther processed. Frozen cells were thawed and 3 mL of cold lysisbuffer (300 mM NaCl, 50 mM NaH₂PO₄, 1% TRITON® X-100, Complete™ ProteaseInhibitor cocktail (Cat. No. sc-29131, Santa Cruz), pH 8) was added toeach cell pellet. The cells were rocked at 4° C. for 30 min followed bysonication (3 pulses for 10 seconds each at 10% power) and finallycentrifuged at 14,000 rpm for 30 min at 4° C. The cleared supernatantwas transferred to a fresh tube and frozen at −80° C. until ready foruse. Prior to capture on an ELISA plate, the supernatant was thawed,briefly centrifuged and diluted 1:2000.

C. Capture with Anti-RSV mAb

ELISA plates were coated using 50 μL/well of a 1:400 dilution ofanti-RSV mAb (Cat. No. NB110-37246, clone 2F7, Novus Biologicals) inPBS. Unbound antibody was removed and the plates were used immediatelyfor ELISA (see Examples 2 and 4). Alternatively, the plates were frozenfor up to 2 weeks at −20° C.

Immediately before use, the plates were blocked with 4% nonfat dry milkin 1×PBS for 2 hours at 37° C. The plates were washed twice with PBScontaining 0.05% TWEEN®-20 (wash buffer) before addition of the lysate.Capture of the RSV F protein (either recombinant or native) was effectedby adding 50 μL of either of the above prepared lysates to each well ofthe anti-RSV mAb ELISA plate and incubating at 37° C. for 2 hours.

Example 2: Isolation of Anti-RSV Fab Antibodies from EBV-Transformed BCells

In this example, anti-RSV antibodies were isolated from stimulatedEpstein Barr virus transformed donor memory B cells, which were screenedfor binding to RSV F protein followed by in vitro antibody generation.Sections A and B describe two slightly different procedures forgenerating and cloning of EBV-transformed B cells and screening forbinding to the RSV F protein. The differences are as follows: (1) thesource of the PMBCs in step 1; (2) isolation of IgG+ B cells in step 2(depleted of IgM, IgD and IgA expressing cells in section A andadditionally depleted of CD3 positive cells in section B); and (3)preparation of irradiated B-cell depleted feeder cells in step 3.a.

A. Generation and Cloning of EBV-Transformed B Cells and Screening forBinding to RSV F Protein (Procedure A)

Peripheral blood mononuclear cells (PBMCs) were obtained from a childcare worker who may have been exposed to RSV through contact withchildren. PBMCs were isolated by density centrifugation over FicollHypaque, according to the manufacturer's instructions

1. CD22+ Isolation and Activation of CD22+ B Cells

3.2×10⁶ CD22+ B cells were isolated from donor PBMCs using CD22 magneticbeads (Miltenyi, cat.#130-046-401) and LS columns (Miltenyi,cat.#130-042-401). Isolated CD22+ B cells were cultured at 1×10⁶ cellsper well in a 48 well plate in RPMI (Hyclone, cat.# SH30096.01)containing 10% heat-inactivated low IgG fetal bovine serum (FBS,Invitrogen, cat.#16250-078), 1% antibiotics (Hyclone, cat.# SV30010), 1%sodium pyruvate (Hyclone, cat.# SH30239.01) and 1% L-glutamine (Hyclone,cat.# SH30034.01). The isolated B cells were activated with a selectionof polyclonal B cell stimulating agents to induce proliferation andantibody production.

2. EBV-Mediated Immortalization of IgG+ B Cells

4.5×10⁶ activated CD22+ B cells were washed and incubated with 40 μL ofFITC-conjugated anti-IgM (BD Biosciences, cat.#555782), 40 μL ofFITC-conjugated anti-IgD (BD Biosciences, cat.#555778) and 4 μL ofFITC-conjugated anti-IgA (Jacksons Immunoresearch, cat. 309-096-043)antibodies for 15 minutes at 4° C. Cells were washed 1× in PBS(containing 0.5% BSA and 2 mM EDTA) and resuspended in 90 uL of the samebuffer. IgG+ B cells were enriched by negative selection of IgM, IgD,IgA expressing cells using 10 μL anti-FITC magnetic beads (Miltenyi,cat.#130-048-701) and LS columns (Miltenyi, cat.#130-042-401) accordingto the manufacturer's instructions.

Bulk immortalization of B cells was performed by incubating 1.87×10⁶IgG+, CD22+ enriched B cells with 0.5 ml EBV supernatant (50% v/v inRMPI-1640 with 10% FCS, ATCC Cat. No. VR-1492 from B95-8 cells) for 16hours. After infection the cells were washed and cultured (10⁶/mL ineach of two wells in RPMI (Hyclone, cat.# SH30096.01) containing 10%heat-inactivated low IgG fetal bovine serum (Invitrogen,cat.#16250-078), 1% antibiotics (Hyclone, cat.# SV30010), 1% sodiumpyruvate (Hyclone, cat.# SH30239.01) and 1% L-glutamine (Hyclone, cat.#SH30034.01), 200 IU/ml rhIL-2 (R&D Systems #202-IL-50) with 0.5×10⁶irradiated feeder cells per well of a 24 well plate for a further 9days.

3. B Cell Cloning

a. Preparation of Irradiated B-Cell Depleted Feeder Cells for Cloning ofB Cells

Irradiated B-cell depleted feeder cells were used to help maintaingrowth of the EBV-transformed B cells. PBMCs from a mixture of 3 healthydonors were obtained by Ficoll separation, irradiated with 3250 rads (atthe UCSD Moore's Cancer Center), and depleted of B cells using anti-CD19magnetic beads (Miltenyi Biotec. Cat. No. 130-050-301) and LD columns(Miltenyi Biotec, cat.#130-091-509). Briefly, frozen and irradiatedPMBCs, obtained from Ficoll separation, were thawed, washed twice andcounted. The cells were then centrifuged at 300 g for 10 minutes, andthe supernatant was aspirated. The cell pellet was resuspended in 80 μlMACS buffer (PBS with 0.5% BSA and 2 mM EDTA) per every 10⁷ cells and 20III CD19 MicroBeads (per every 10⁷ cells) was added. Following thoroughmixing, the cells were incubated at 4° C. for 15 minutes. The cells werethen washed by adding 1-2 mL buffer (per every 10⁷ cells) followed bycentrifuging at 300 g for 10 minutes and the supernatant was aspirated.Up to 10⁸ cells were then resuspended in 500 μl buffer. Magneticseparation was effected by placing a LD column (composed offerromagnetic spheres covered with a plastic coating to allow fast andgentle separation of cells) in the magnetic field of a MACS separator.The LD column was washed with 2 mL buffer and the cell suspension wasapplied to the top of the column. Non-B cells were collected as theypassed through the column after the addition of 2×1 mL buffer.

b. B Cell Cloning

Approximately 20 EBV-transformed B-cells were co-cultured withpolyclonal B cell stimulating agents and 50,000 irradiated B-celldepleted feeder cells per well in a 96 well plate and grown for 13 days.A total of 120 96-well plates were generated.

4. Screening of B Cell Supernatant for Binding to RSV F Protein

Supernatants from each well were transferred to a new 96-well plate andthe cells were washed 1× in PBS and frozen at −80° C. in 100 μL of RLTbuffer (Qiagen, cat.#79216) containing 10 μL/mL 2-mercaptoethanol. Thesupernatant was used in an ELISA to determine which wells are producingantibodies that are capable of binding to RSV F protein. Briefly, theELISA was performed as follows: (I) RSV F Protein ELISA plates wereprepared as described in Example 1 using 96 well half-area plates withthe following modifications: anti-RSV mAb (clone 2F7, mouse ascitesfluid, Cat. No. ab43812, Abcam) was used as the capture antibody and theRSV F protein was incubated with the capture antibody overnight at 4° C.(2) 10 μL B cell supernatant from each of 2 wells (a total of 20 μLpooled) was added to a 96 half-well ELISA plate and incubated for 2 h at37° C. Plasma from a pool of Blood Bank donors (collected and frozenafter Ficoll Hypaque separation, diluted 1:1000) was used as a positivecontrol. (3) Plates were washed 4× as above and 50 μL of goat anti-humanFc IgG HRP-conjugated antibody (diluted 1:1000 in PBS with 0.05%TWEEN®20) was added to each well and the plate was incubated at 37° C.for 1 hour. (4) Plates were washed 6× as above and developed using 50 μLof 1:1 v/v TMB:peroxide solution (Pierce, Cat No. 34021) substrate andallowed to develop for 7 minutes. The reaction was immediately halted bythe addition of 50 μL 2N H₂SO₄ and the absorbance at 450 nm was measuredusing an ELISA plate reader. Positive binding was indicated by an OD₄₅₀greater than 0.5 (0.5-0.9 is moderate binding, >1 is strong binding) anda response that was 3-fold above background.

To determine which of the two pooled wells contained anti-RSVantibodies, 20 μL of B cell supernatant (diluted 1:2 v/v with PBS/0.05%TWEEN® 20) from each well was retested individually against captured RSVF protein.

A total of 18 plates (or 1080 wells) were screened for binding to RSV Flysate (as purified in Example 1). Six wells were identified as bindersto RSV F lysate. Five of the six wells were reconfirmed by an additionalELISA and used to generate anti-RSV antibodies by PCR (described below).

B. Generation and Cloning of EBV-Transformed B Cells and Screening forBinding to RSV F Protein (Procedure B)

Peripheral blood mononuclear cells (PMBCs) were obtained from a SanDiego Blood Bank donor. PBMCs were isolated by density centrifugationover Ficoll Hypaque, according to the manufacturer's instructions.

1. CD22+ Isolation and Activation of CD22+ B Cells

10.5×10⁶ CD22+ B cells were isolated from donor PBMCs using CD22magnetic beads and the isolated CD22+ B cells were cultured andactivated in 24 well plates, as described in Section A.1. above.

2. EBV-Mediated Immortalization of IgG+ B Cells

a. Isolation of IgG+ B Cells

11.3×10⁶ activated CD22+ B cells were washed and incubated with 110 μLof biotin-conjugated anti-IgM (BD Biosciences, cat. #555781), 22 μL ofbiotin-conjugated anti-IgD (BD Biosciences, cat. #555777) and 22 μL ofbiotin-conjugated anti-IgA (Invitrogen, cat. #62-7440) and 220 μL ofbiotin-conjugated anti-CD3 (BD Bioscience, cat. #555331) antibodies for15 minutes at 4° C. Cells were washed 1× in PBS (containing 0.5% BSA and2 mM EDTA) and resuspended in 1.045 mL of the same buffer. IgG+ B cellswere enriched by negative selection of IgM, IgD, IgA and CD3 expressingcells using 55 μL anti-biotin magnetic beads (Miltenyi,cat.#130-090-485) and LD columns (Miltenyi, cat.#130-042-901) accordingto the manufacturer's instructions.

b. Preparation of Irradiated Feeder Cells for Culture of EBV TransformedCells

Irradiated feeder cells were used to help maintain growth of theEBV-transformed B cells. PBMCs from a mixture of 2-3 healthy donors wereobtained and isolated by Ficoll separation, irradiated with 3300 rads(at the UCSD Moore's Cancer Center) and frozen at 5×10⁶ cells per vialin FBS containing 10% dimethylsulfoxide (Sigma cat. # D2650-100), Beforeuse, one vial of cells was thawed and washed 3× with RPMI (Hyclone,cat.# SH30096.01) containing 10% heat-inactivated low IgG fetal bovineserum (Invitrogen, cat. #16250-078), 1% antibiotics (Hyclone, cat. #SV30010), 1% sodium pyruvate (Hyclone, cat.# SH30239.01) and 1%L-glutamine (Hyclone, cat. # SH30034.01). The cells were counted,resuspended at 1×10⁶ cells/ml and plated at 0.25×10⁶ cells per well of a48 well plate.

c. EBV-Mediated Immortalization

Bulk immortalization of B cells was performed by incubating 0.58×10⁶IgG+, CD22+ enriched B cells with 0.5 ml EBV supernatant (50% v/v inRMPI-1640 with 10% FCS, ATCC Cat. No. VR-1492 from B95-8 cells) for 16hours. After infection the cells were washed and cultured (0.58×10⁶/0.5mL in one well in RPMI (Hyclone, cat.# SH30096.01) containing 10%heat-inactivated low 12G fetal bovine serum (Invitrogen,cat.#16250-078), 1% antibiotics (Hyclone, cat.# SV30010), 1% sodiumpyruvate (Hyclone, cat. # SH30239.01) and 1% L-glutamine (Hyclone, cat.#SH30034.01) with 0.5×10⁶ irradiated feeder cells per well of a 48 wellplate. After 48 hours, 200 IU/ml rhIL-2 (R&D Systems, cat. #202-IL-50)was added and the cells were cultured for a further 7 days.

3. B Cell Cloning

a. Preparation of Irradiated B-Cell Depleted Feeder Cells for Cloning ofB Cells

Irradiated B-cell depleted feeder cells were prepared for cloning of Bcells. Buffy coats from 5-6 healthy donors were obtained and storedovernight at ambient temperature. The PBMCs were isolated by Ficollseparation, and depleted of B cells using anti-CD19 magnetic beads(Miltenyi Biotec, Cat. No. 130-050-301) and LD columns (Miltenyi Biotec,cat. #130-042-901).

Briefly, frozen PMBCs, obtained from Ficoll separation, were thawed,washed twice and counted. The cells were then centrifuged at 300 g for10 minutes, and the supernatant was aspirated. The cell pellet wasresuspended in 80 μl MACS buffer (PBS with 0.5% BSA and 2 mM EDTA) perevery 10⁷ cells and 20 μl CD19 MicroBeads (per every 10⁷ cells) wasadded. Following thorough mixing, the cells were incubated at 4° C. for15 minutes. The cells were then washed by adding 1-2 mL buffer (perevery 10⁷ cells) followed by centrifuging at 300 g for 10 minutes andthe supernatant was aspirated. Up to 10⁸ cells were then resuspended in500 μl buffer. Magnetic separation was effected by placing a LD column(composed of ferromagnetic spheres covered with a plastic coating toallow fast and gentle separation of cells) in the magnetic field of aMACS separator. The LD column was washed with 2 mL buffer and the cellsuspension was applied to the top of the column. Non-B cells werecollected as they passed through the column after the addition of 2×1 mLbuffer. The cells were centrifuged at 300 g for 10 minutes andresuspended at 5×10⁶ cells/mL in PBS containing 2% low IgG fetal calfserum. The cells were irradiated with 4000 rads (at the UCSD Moore'sCancer Center), centrifuged at 300 g for 6 minutes, resuspended in 90%low IgG FBS and 10% dimethylsulfoxide (DMSO, 25×10⁶ cells/mL) and storedfrozen in liquid nitrogen.

Before use, the cells were thawed and washed three times with Iscove'sModified Dulbeccos Medium (IMDM, Hyclone #SH30228.01) containing 10%heat-inactivated low IgG fetal bovine serum (Invitrogen, cat.#16250-078), 1% antibiotics (Hyclone, cat. # SV30010), 1% nonessentialamino acids (Hyclone, cat. # SH30238.01) and 1% L-glutamine (Hyclone,cat. # SH30034.01).

b. B Cell Cloning

Approximately 100 EBV-transformed B-cells were co-cultured withpolyclonal B cell stimulating agents and 50,000 irradiated B-celldepleted feeder cells per well in a 96 well plate and grown for 13 daysin IMDM (Hyclone, cat. # SH30228.01) containing 10% heat-inactivated lowIgG fetal bovine serum (Invitrogen, cat. #16250-078), 1% antibiotics(Hyclone, cat. # SV30010), 1% nonessential amino acids (Hyclone, cat.#SH30238.01) and 1% L-glutamine (Hyclone, cat. # SH30034.01). A total of120 96-well plates were generated.

4. Screening of B Cell Supernatant for Binding to RSV F Protein

Supernatant from each well was screened by ELISA to determine whichwells were producing antibodies that are capable of binding to RSV Fprotein. The ELISA was performed as described in Section A.5. above in96-well half-area ELISA plates coated with RSV F protein (as describedin Example 1). As described above, to determine which of the two pooledwells contained anti-RSV antibodies, 20 μL of B cell supernatant(diluted 1:2 v/v with PBS/0.05% TWEEN® 20) from each well was retestedindividually against captured RSV F protein.

A total of 120 plates (or 7200 wells) were screened for binding to RSV Flysate (as purified in Example 1). Twenty nine wells were identified asbinders to RSV F lysate. Ten of the twenty five wells were reconfirmedby an additional ELISA and used to generate anti-RSV antibodies by PCR(described below).

C. Generation of Anti-RSV Antibodies by PCR

Following initial screening of EBV-transformed B cells for production ofantibodies that bind to RSV F protein, genes encoding individualantibodies were amplified from B cell RNA by PCR. Five wells identifiedas hits from Section A above were selected for cloning. Ten wellsidentified as hits in Section B above were selected for cloning.

1. RNA Extraction

RNA was extracted from the B cells (for each well corresponding to apositive binder to RSV F protein) using an RNeasy Micro Kit (Qiagen,Cat. No. 1402-2408) according to the manufacturer's instructions withthe following modifications: 1) B cells were frozen in 100 μL RLT bufferwith β-mercaptoethanol (10 μL per mL buffer); 2) the cells were nothomogenized; 3) RNA was precipitated with 70% ethanol (in RNase-freewater); and 4) DNase treatment was carried out “in-column” according themanufacturer's supplemental protocol. The RNA was eluted into a finalvolume of 26 μL.

2. First Strand cDNA Synthesis

Following RNA extraction, cDNA was generated according to theSuperscript III (Invitrogen; Cat No. 19090-051) First Strand Synthesisprotocol. Briefly, 8 μl RNA (isolated as described above), 1 μl oligo dTprimer and 1 μl dNTPs were combined in a sterile 0.2 mL tube andincubated at 65° C. for 5 minutes followed by incubation on ice for 1minute. Subsequently, 2 μl 0.1 mM DTT, 4 μl 25 mM MgCl₂ 2 μl RT buffer,1 μl RNaseOut, and 1 μl SuperScript III RT were added to the tube, andthe reaction mixture was incubated at 50° C. for 50 minutes followed byincubation at 85° C. for 15 minutes. The cDNA was used immediately orfrozen at −80° C. for long term storage.

3. Isolation of IgG Heavy Chain and Kappa and Lambda Light Chain Genesby PCR Amplification

IgG heavy chains and kappa and lambda light chains were generated by PCRamplification from the B cell first strand cDNA synthesis reaction (seeabove). The kappa light chain genes were amplified by a single-step PCR,whereas the heavy chain genes and lambda light chain genes wereamplified using a two-step, nested PCR approach. The amplified heavy andlight chain genes were subsequently linked into a single cassette using“overlap PCR”.

Step I. Amplification or IgG Heavy Chain Genes and Lambda Light ChainGenes

In Step I, Heavy Chain A, 2 μL cDNA generated by First Strand Synthesis(see above) was used as a template to individually amplify IgG heavychains by PCR. In this step, pools of Step I primers were utilized (seeTable 3A below). The reaction conditions were as follows:

PCR Step I: Heavy Chain A:

Reagent μL H₂O 16 10x buffer 2.5 10x Enhancer buffer 2.5 dNTP (10 mMeach) 0.75 cDNA 2.0 VH pool leader (9 μM each) 0.5 VH Reverse pool (20μM) 0.25 Pfx50 0.5 25

Alternatively, in Step I, Heavy Chain B, 2.5 μL cDNA generated by FirstStrand Synthesis (see above) was used as a template to individuallyamplify IgG heavy chains by PCR. In this step, the same pool of Step Iprimers was utilized for the forward primers and VH gamma-1(a/b) reversewas used as the reverse primer (see Table 3A below). The reactionconditions were as follows:

PCR Step I: Heavy Chain B:

Reagent μL H₂O 15.5 10x buffer 2.5 10x Enhancer buffer 2.5 dNTP (10 mMeach) 0.75 cDNA 2.5 VH pool leader (9 μM each) 0.5 VH-g1(a/b)-REV (20μM) 0.25 Pfx50 0.5 25

In Step I, Lambda Light Chain, 2.5 μL cDNA generated by First StrandSynthesis (see above) was used as a template to individually amplify IgGheavy chains by PCR. In this step, a pool of Step I primers was utilizedfor the forward primers and pCALCL(T)-R was used as the reverse primer(see Table 3B below). The reaction conditions were as follows:

PCR Step I: Lambda Light Chain:

Reagent μL H₂O 16 10x buffer 2.5 10x Enhancer buffer 2.5 dNTP (10 mMeach) 0.75 cDNA 2.0 Vλ pool (14.2 μM each) 0.5 pCALCL(T)-R (20 μM) 0.25Pfx50 0.5 25

For the PCR reaction, a touchdown approach was implemented in order toadd specificity to the reaction amplification. At each touchdown step,the annealing temperature is decreased by 1° C. every cycle. The PCRthermocycler conditions were as follows.

1) 94° C. for 2 minutes

2) 10 cycles of:

-   -   94° C. for 15 seconds; 62° C. for 20 seconds (Touchdown); 68° C.        for 1 minute

3) 25 cycles of:

-   -   94° C. for 15 seconds; 52° C. for 20 seconds; 68° C. for 1        minute

4) 68° C. for 3 minutes

5) 4° C. hold

The resultant reaction mixtures were used as template DNA for Step II(see below) without any further purification.

TABLE 3A Step I Primers for Amplifying IgG Heavy Chain Genes SEQVH Forward Primer Pool: ID NO: VH1a GGATCCTCTTCTTGGTGGCAGCAG 26 VH1bGCATCCTTTTCTTGGTGGCAGCAC 27 VH1c GGGTCTTCTGCTTGCTGGCTGTAG 28 VH1dGGATCCTCTTCTTGGTGGCAGCAG 29 VH2a CTGACCATCCCTTCATGGCTCTTG 30 VH2bCTGACCACCCCTTCCTGGGTCTTG 31 VH3a GCTATTTTARAAGGTGTCCAGTGT 32 VH3bGCTCTTTTAAGAGGTGTCCAGTGT 33 VH3c GCTATTTAAAAAGGTGTCCAATGT 34 VH4aCTGGTGGCAGCTCCCAGATGGGTC 35 VH5a CTCCTGGCTGTTCTCCAAGGAGTC 36 SEQVH Reverse Primer Pool: ID NO: VH-g 1-REV ACAAGATTTGGGCTCAACTTTCTTGTCC37 VH-g 2-REV TTTGCGCTCAACTGTCTTGTCCACCTTG 38 VH-g 3-REVTTTGAGCTCAACTCTCTTGTCCACCTTG 39 VH-g 4-REV ATATTTGGACTCAACTCTCTTGTCCACC40 SEQ Reverse Primer: ID NO: VH-g1(a/b)-REVACAAGATTTGGGCTCAACTYTCTTGTCC 383

TABLE 3B Step I Primers for Amplifying Lambda Light Chain GenesForward Primer Pool: SEQ ID NO: 5′ L Vλ 1 GGTCCTGGGCCCAGTCTGTGCTG 384 5′L Vλ 2 GGTCCTGGGCCCAGTCTGCCCTG 385 5′ L Vλ 3 GCTCTGTGACCTCCTATGAGCTG 3865′ L Vλ 4/5 GGTCTCTCTCSCAGCYTGTGCTG 387 5′ L Vλ 6GTTCTTGGGCCAATTTTATGCTG 388 5′ L Vλ 7 GGTCCAATTCYCAGGCTGTGGTG 389 5′L Vλ 8 GAGTGGATTCTCAGACTGTGGTG 390 Reverse Primer: SEQ ID NO:pCALCL(T)-R CTCCTTATTAATTAATTATGAGCATTC 80 TGYAKGGGCMAYTGTC

Step II. Amplification of Heavy and Light Chain Genes

In Step II, the heavy chain and lambda light chain reaction mixturesfrom Step I were used as templates for second round PCR reactions withpools of forward and reverse primers that amplify from the framework 1region of each chain to the end of the constant region (C_(H)1 for heavychain, C_(L) for light chain).

The heavy chain forward primers (see Table 4) were designed to introducea SfiI restriction site (SEQ ID NO:41). The reaction conditions were asfollows:

PCR II: Heavy Chain

Reagent μL H₂O 12.75 10x buffer 2.5 10X Enhancer 2.5 dNTP (10 mM each)0.75 Step I reaction 2.5 pCAL24VH-F pool (2 μM) 2.5 CH1-R Pool-Sfi (20μM) 1 Pfx50 0.5 25

The lambda light chain forward primers (see Table 6) were designed tointroduce a SfiI restriction site (SEQ ID NO:41). The reactionconditions were as follows:

PCR II: Lambda Light Chain

Reagent μL H₂O 15.5 10x buffer 2.5 10X Enhancer 2.5 dNTP (10 mM each)0.75 Step I reaction 2.5 Vλ primer pool (2 μM) 2.5 pCALCL(T)R (20 μM) 1Pfx50 0.5 25

The PCR thermocycler conditions for Step II reactions were as follows:

1) 94° C. for 2 minutes

2) 30 cycles of:

-   -   94° C. for 15 seconds; 52° C. for 20 seconds; 68° C. for 1        minute

3) 68° C. for 3 minutes

4) 4° C. hold

For amplification of kappa light chain genes, 2 μL cDNA generated byFirst Strand Synthesis (see above) was used as a template toindividually amplify IgG kappa light chains by PCR. The light chainkappa forward primers (see Table 5) were used as primer pools and weredesigned to introduce a SfiI restriction site (SEQ ID NO:41).

The reaction conditions were as follows:

PCR II: Kappa Light Chain

Reagent μL H₂O 16 10x buffer 2.5 10X Enhancer 2.5 dNTP (10 mM each) 0.75First Strand cDNA 2 Vκ Primer Pool (9.1 μM) 0.5 pCALCK(G)L (20 μM) 0.25Pfx50 0.50 25

The PCR thermocycler conditions for Step II reactions were as follows:

1) 94° C. fort minutes

2) 35 cycles of:

-   -   94° C. for 15 seconds; 54° C. for 20 seconds: 68° C. for 1        minute

3) 8° C. for 3 minutes

4° C. hold

Following amplification, the PCR reaction products were separated on a1% agarose gel and the band corresponding to the heavy chain (675 bp)and the light chain (650 bp) were purified by gel extraction (Qiagen GelExtraction Kit; Cat. No. 28706). The PCR products were eluted in 30 μl.

TABLE 4 Primers for Amplifying IgG Heavy Chain Genes Forward Primer PoolSEQ ID NO: pCal30 VH1a ggctttgctaccgtagcgCAGGCGGCCGCACAGGTKCAGCTGGTGCAG42 pCal30 VH1b ggctttgctaccgtagcgCAGGCGGCCGCACAGGTCCAGCTTGTGCAG 43pCal30 VH1c ggctttgctaccgtagcgCAGGCGGCCGCASAGGTCCAGCTGGTACAG 44pCal30 VH1d ggctttgctaccgtagcgCAGGCGGCCGCACARATGCAGCTGGTGCAG 45pCal30 VH2a ggctttgctaccgtagcgCAGGCGGCCGCACAGATCACCTTGAAGGAG 46pCal30 VH3a ggctttgctaccgtagcgCAGGCGGCCGCAGARGTGCAGCTGGTGGAG 47pCal30 VH4a ggctttgctaccgtagcgCAGGCGGCCGCACAGSTGCAGCTGCAGGAG 48pCal30 VH4b ggctttgctaccgtagcgCAGGCGGCCGCACAGGTGCAGCTACAGCAG 49pCal30 VH5a ggctttgctaccgtagcgCAGGCGGCCGCAGARGTGCAGCTGGTGCAG 50pCal30 VH6 ggctttgctaccgtagcgCAGGCGGCCGCACAGGTACAGCTGCAGCAG 51pCal30 VH7 ggctttgctaccgtagcgCAGGCGGCCGCACAGGTSCAGCTGGTGCAA 52Reverse Primer Pool SEQ ID NO: VHII-g1-RevTGCGGCCGGCCTGGCCGACCACAAGATTTGGGCTCAACTTTC 53 VHII-g2-RevTGCGGCCGGCCTGGCCGACCTTTGCGCTCAACTGTCTTGTCC 54 VHII-g3-RevTGCGGCCGGCCTGGCCGACCTTTGAGCTCAACTCTCTTGTCC 55 VHII-g4-RevTGCGGCCGGCCTGGCCGACCATATTTGGACTCAACTCTCTTG 56

TABLE 5 Primers for Amplifying Kappa Light Chain GenesForward Primer Pool SEQ ID NO: VK1aAAggcccagccggccatggccgccggtGACATCCAGATGACCCAG 57 VK1bAAggcccagccggccatggccgccggtGACATCCAGTTGACCCAG 58 VK1cAAggcccagccggccatggccgccggtGCCATCCGGTTGACCCAG 59 VK2aAAggcccagccggccatggccgccggtGATATTGTGATGACYCAG 60 VK3aAAggcccagccggccatggccgccggtGAAATTGTGTTGACGCAG 61 VK3bAAggcccagccggccatggccgccggtGAAATTGTGTTGACACAG 62 VK3cAAggcccagccggccatggccgccggtGAAATAGTGATGACGCAG 63 VK4aAAggcccagccggccatggccgccggtGACATCGTGATGACCCAG 64 VK5aAAggcccagccggccatggccgccggtGAAACGACACTCACGCAG 65 VK6aAAggcccagccggccatggccgccggtGAAATTGTGCTGACTCAG 66 VK6bAAggcccagccggccatggccgccggtGATGTTGTGATGACACAG 67 Reverse PrimerSEQ ID NO: pCALCK (G) L CTCCTTATTAATTAATTAGCACTCTCCCCTGTTGAAGCTCTTTG 68

TABLE 6 Primers for Amplifying Lambda Light Chain GenesForward Primer Pool SEQ ID NO: VL1-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTCAGTCTGTGCTGACKCAGCC 69 VL2-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTCAGTCTGCCCTGACTCAGCC 70 VL3A-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTTCCTATGAGCTGACWCAGCY 71 VL3B-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTTCTTCTGAGCTGACTCAGGAC 72 VL3C-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTTCCTATGWGCTGACTCAGCC 73 VL4A-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTCTGCCTGTGCTGACTCAGCCC 74 VL4B-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTCAGCYTGTGCTGACTCAATCR 75 VL5/9-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTCAGSCTGTGCTGACTCAGCCR 76 VL6-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTAATTTTATGCTGACTCAGCCC 77 VL7/8-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTCAGRCTGTGGTGACTCAGGAG 78 VL10-FAAGGCCCAGCCGGCCATGGCCGCCGGTGTTCAGGCAGGGCTGACTCAGCCA 79 Reverse PrimerSEQ ID NO: pCALCL(T)-R CTCCTTATTAATTAATTATGAGCATTCTGYAKGGGCMAYTGTC 80

Step III. Overlap PCR

In Step III, the heavy chain and light chain DNA segments generated inStep II were 1) linked in an overlap reaction with a Fab linker (seeTable 7, below) that anneals to the 3′ end of the light chain and the 5′end of the heavy chain and 2) amplified with a SE forward and reverseprimers (see Table 7, below), thereby allowing amplification of a 1200base pair (bp) antibody fragment containing the light chain-linker-heavychain.

The Fab Kappa Linker was amplified from the 2gI2/pCAL vector (SEQ IDNO:81) using either the FabLinker-Rev or the FabLinker-Rev-IT* primers(see Table 7 below). The PCR reaction conditions for the formation ofthe Fab Kappa Linker were as follows:

Fab Kappa Linker

Reagent μL H₂O 19.75 10x buffer 2.5 dNTP (10 mM each) 0.75 2g12/pCALVector (10 ng) 1 FabLinkerCK-Fwd (20 μM) 0.25 Reverse Primer (20 μM)0.25 Pfx50 0.5 25

The Fab Lambda Linker was amplified from the 28d11/pCAL vector (SEQ IDNO:1636). The PCR reaction conditions for the formation of the Fab KappaLinker were as follows:

Fab Lambda Linker

Reagent μL H₂O 35.5 10x buffer 5 10x enhancer 5 dNTP (10 mM each) 1.528d11/pCAL Vector (10 ng) 1 FabLinkerCλ-Fwd (20 μM) 0.5 FabLinker-RevIT* (20 μM) 0.5 Pfx50 0.5 25

The PCR thermocycler conditions for the formation of the Fab Linkerswere as follows:

1) 94° C. for 2 minutes

2) 30 cycles of:

-   -   94° C. for 15 seconds; 54° C. for 20 seconds; 68° C. for 1        minute

3) 68° C. for 3 minutes

4) 4° C. hold

The PCR reaction was run on a 1% agarose gel and the 120 bp linker wasgel extracted according to the Qiagen Gel Extraction protocol. 2 μl ofthe purified linker was used for each overlap reaction.

The PCR reaction conditions for Overlap were as follows (the Sfi F/RPrimers are added to the PCR reaction after the first 15 cycles):

PCR III: Overlap

Reagent μL H₂O 24.5 10x buffer 5 10X Enhancer 5 dNTP (10 mM each) 1.5Light Chain product 5 Heavy Chain product 5 Linker 2 Sfi F/R Primers (20μM) 1 Pfx50 1 50

The PCR thermocycler conditions were as follows:

1) 94° C. for 2 minutes

2) 15 cycles of:

-   -   94° C. for 15 seconds: 68° C. for 1 minute;

Add Sfi F/R Primers (1 μL), then:

3) 94° C. for 2 minutes

4) 30 cycles of:

-   -   94° C. for 15 seconds; 60° C. for 20 seconds; 68° C. for 2        minutes

5) 68° C. for 3 minutes

6) 4° C. hold

Following amplification, 10 μl of the total 50 μl PCR overlap reactionproduct light chain-linker-heavy chain was separated on a 1% agarose gelto determine the size and the remaining 40 μl of the PCR product waspurified by the Qiagen PCR Purification Kit (Qiagen; Cat. No. 28106)into 30 μl total volume. Briefly, 5 times the PCR reaction volume of PBIbuffer is added PCR product. The mix was bound to QIA Spin column andwashed twice with PE buffer. The sample was eluted in 30 μl and spun for1.5 minutes at top speed to elute all 30 μl. About 1 μg of overlapproduct was the typical yield per 50 μl overlap reaction.

TABLE 7 Step III Oligonucleotides SEQ ID Oligonucleotide NO:FabLinkerCK-Fwd GAGCTTCAACAGGGGAGAGTGCTAATT 82 AATTAATAAGGAGFabLinker-Rev TGCGGCCGCCTGCGCTACGGTAGCAAAG 83 GCCACCAGTGCCACFabLinkerCλ-Fwd GACARTKGCCCMTRCAGAATGCTCATA 392ATTAATTAATAAGGAGGATATAATTATG AAAAAG FabLinker-Rev-TGCGGCCGCCTACGCTACGGTAGCAAAG 393 IT* CCAGCCAGTGCCAC Sfi ForwardTCGCggcccagccggccatggc 84 Sfi Reverse TGCGGCCGGCCTGGCCGA 85

Step IV. Digestion with Sfi and Cloning into Either the pCAL or pCAL IT*Expression Vector

Following overlap PCR reaction and purification of the PCR product, thereaction product was digested with SfiI. To the 30 μl eluate (seeabove), the following was added for the digestion:

 4 μl Reaction buffer 2 (New England Biolabs) 0.4 μl BSA 1.6 μl SfiIenzyme (New England Biolabs)  4 μl H2O  40 μl Total Volume

The reaction is incubated for 1 hour at 37° C. Following digestion, thedigested overlap product was separated on a 1% agarose gel and the bandcorresponding to the antibody (˜1.45 kB) was purified by gel extraction(Qiagen, Gel Extraction Purification Kit Cat. No. 28706). Briefly, thegel slice was digested with 500 μl of buffer QC (Qiagen). 150 μl ofisopropanol was added to digest and the sample was applied to theQiaSpin column. The column was washed twice with buffer PE (Qiagen) andthe sample is eluted in 30 μl of EB buffer (Qiagen). About 15 ng/μl ofdigested sample is recovered from approximately 1 μg of PCR overlapproduct.

Finally, the digested overlap product was ligated into the pCAL (SEQ IDNO:86) or the pCAL IT* (SEQ ID NO:394) bacterial expression vector. Theligation reaction conditions were as follows:

25 ng SfiI digested pCAL or pCAL IT* vector 25 ng digested overlapproduct 2 μl T4 Ligase reaction buffer 1 μl T4 Ligase (NEB Cat. No.MC202L, 400,000 Units/ml) adjusted to 20 μl total volume with H₂O

The sample was ligated for 1 hour at room temperature. 1 μl of theligation was diluted in 4 μl of H₂O before proceeding to transformation.

Step V. Transformation into E. coli

Following ligation, the ligation product was transformed into DH5α MaxEfficiency cells (Invitrogen; Cat No. 18258; Genotype: F− φ80lacZΔM15Δ(lacZY A-argF) U169 recA1 endA1 hsdR17 (rk−, mk+) phoA supE44 λ− thi-1gyrA96 relA1). In short, 1 μl ligation product (1/5 dilution) was addedto 50 μl DH5α and incubated on ice for 30 minutes. Transformation waseffected by heat shock at 42° C. for 45 seconds followed by 2 minutes onice. 0.9 mL SOC medium was added and the cells were allowed to recoverat 37° C. for 1 hour with shaking. Cells were plated on LB platessupplemented with carbenicillin (100 μg/mL) and 20 mM glucose. Theplates were incubated overnight at 37° C.

Step VI. Selection of Individual Colonies.

For each antibody amplification, a total of 88 individual colonies wereselected and grown in 1 mL Super Broth (SB) supplemented with 1carbenicillin (100 μg/mL) in a 96-well plate for 2 hours at 37° C. Adaughter plate was generated by transferring 500 μl of each culture intoanother 96-well format bacterial plate with 500 μl of SB supplementedwith 40 mM glucose (final 20 mM) and 100 μg/ml of carbenicillin. Theoriginal or mother plate was fed 500 μl of SB supplemented with 100ug/ml carbenicillin. The original plate was grown at 30° C. overnightand the daughter plate (containing glucose) was grown at 37° C.overnight. The cell lysate from the 30° C. plate was used for bacterialELISAs (see Example 4 below) and the 37° C. plate cultures were used formini-prep DNA preparations (Qiagen).

Summary

The five wells identified as hits from Section A above were amplifiedusing kappa light chain primers and cloned into the pCAL expressionvector. The ten wells identified as hits in Section B above wereamplified with kappa light chain primers and cloned into the pCAL IT*expression vector. One well was additionally amplified with lambda lightchain primers and cloned into the pCAL IT* expression vector.

Example 3: Isolation of Anti-RSV Fab Antibodies by Single Cell Sorting

In this example, anti-RSV antibodies were isolated from CD19/CD27/IgGpositive cells. The CD19/CD27/IgG positive cells were obtained by 1) Bcell isolation; and 2) FACS single cell sorting. The sorted cells werethen used to isolate RNA which served as a template for the in vitroproduction of Fab antibodies.

B Cell Isolation

B cells were isolated from PBMCs (harvested from an anonymous blood bankdonor) using a B Cell Isolation Kit (Miltenyi Biotec, Cat. No.130-091-151). The kit is used to isolate highly pure B cells by magneticlabeling and depletion of CD2, CD14, CD16, CD36, CD43, andCD235a-expressing cells (activated B cells, plasma cells and CD5⁺ B-1acells) and non-B cells (e.g., T cells, NK cells, dendritic cells,macrophages, granulocytes, and erythroid cells). According to themanufacturer's protocol, non-B cells were indirectly magneticallylabeled by using a cocktail of biotin-conjugated monoclonal antibodiesas a primary labeling reagent (Biotin-Antibody Cocktail) and anti-biotinmonoclonal antibody conjugated to microbeads as a secondary labelingreagent (Anti-Biotin MicroBeads). The non-B cells were then removed fromthe pure resting B cells by magnetic separation.

Briefly, frozen PMBCs, obtained from Ficoll separation, were thawed,washed twice and counted. The cells were then centrifuged at 300 g for10 minutes, and the supernatant was aspirated. The cell pellet wasresuspended in 40 μl MACS buffer (per every 10⁷ cells) and 10 μlBiotin-Antibody Cocktail (per every 10⁷ cells) was added. Followingthorough mixing, the cells were incubated at 4° C. for 10 minutes. Afterthe incubation period, 30 μl buffer (per every 10⁷ cells) and 20 μlAnti-Biotin MicroBeads (per every 10⁷ cells) was added. Followingthorough mixing, the cells were incubated at 4° C. for 15 minutes. Thecells were then washed by adding 1-2 mL buffer (per every 10⁷ cells)followed by centrifuging at 300 g for 10 minutes and the supernatant wasaspirated. Up to 10⁸ cells were then resuspended in 500 μl buffer.

Magnetic separation was effected by placing a LS column (composed offerromagnetic spheres covered with a plastic coating to allow fast andgentle separation of cells) in the magnetic field of a MACS separator.The LS column was washed with 3 mL buffer and the cell suspension wasapplied to the top of the column. Unlabeled B cells were collected asthey passed through the column after the addition of 3×3 mL buffer.

Single Cell Sorting

In this example, isolated B cells were sorted by antigen specificityusing an FACSAria Flow Cytometer (BD Biosciences). Selected cells wereCD19/CD27/IgG positive. RSV-F antigen was labeled with Alexa Fluor 647following the manufacturer's instructions (Molecular Probes, A-20186).

In short, the isolated B cells were aliquotted into 16 separate tubes.Fourteen tubes received 1×10⁸ cells and were used to determine thephotomultiplier settings and sort parameters on the FACSAria. Theremaining 1.8×10⁶ cells were labeled with Alexa Fluor 647/RSV-F at afinal concentration of 20 nM. Labeled protein was added to the sample 15minutes prior to the addition of antibodies. CD19 and CD27 antibodieswere used at dilution of 1:20 while IgG antibody was used at a dilutionof 1:50. Following the addition of Alexa Fluor 647/RSV-F protein andantibodies, the tubes were incubated on ice for 30 minutes andsubsequently washed twice. Single cell sorting was effected using theFACSAria Flow Cytometer (BD Biosciences). The labels included PE-Cy5(anti-human CD19), PE-Cy7 (anti-human CD27), PE (goat anti-human IgGFcg), Pacific Blue (mouse anti-human CD3), FITC (mouse anti-human IgD,mouse anti-human IgM, mouse anti-human IgA and mouse anti-human CD14),propidium iodide and Alexa Fluor 647 (labeled RSV-F protein).

Cell sorting was performed by first excluding dead cells followed byexclusion of CD3 positive cells. CD19 and CD27 positive cells werefurther identified and within this population, cells were gated for IgGFcγ expression. Cells expressing IgD, IgM and IgA were excluded from theremaining cells. Finally, CD19/CD27/IgG Fcγ positive cells were sortedfor RSV-F binding and each positive B cell was deposited into anindividual well of a 96 well plate containing 2 μl cDNA reaction buffer(Superscript III 10× buffer. Invitrogen; Cat No. 19090-051), 0.5 μlRNaseOUT and 7.5 μl sterile water. Plates were stored at −80° C. untilfurther processed.

First Strand cDNA Synthesis

Following sorting, cDNA was generated individually in each wellaccording to the Invitrogen First Strand Synthesis protocol. In short,0.5 μl 10% NP-40, 1 μl oligo dT primer and 1 μl dNTPs were added to eachwell and the plate was incubated at 65° C. for 5 minutes followed byincubation on ice for 1 minute. Subsequently, 2 μl DTT, 4 μl MgCl₂ and 1μl SuperScript III RT were added and the reaction mixture was incubatedat 50° C. for 1 hour followed by incubation at 85° C. for 5 minutes. ThecDNA was used immediately or frozen at −80° C. for long term storage.

IgG Heavy Chain and Kappa Light Chain Amplification

IgG heavy chains and kappa light chains were subsequently generated byfour sequential steps of PCR.

Step I. Amplification

In Step I, 2.5 μL cDNA generated by First Strand Synthesis (see above)was used as a template to individually amplify kappa light chains andIgG heavy chains by PCR. In this step, pools of Step I primers wereutilized (see Tables 8 and 9 below). The reaction conditions were asfollows:

PCR Step I:

H₂O 16 10x buffer 2.5 10x Enhancer buffer 2.5 dNTP (10 mM each) 0.75cDNA 2.5 Step I pool(20 μM each) 0.25 Reverse Primer (20 μM) 0.25 Pfx500.25 25 μL

The PCR thermocycler conditions were as follows:

1) 94° C. for 2:00

2) 10 cycles of:

-   -   94° C. for 0:15; 62° C. for 0:20 (TOUCHDOWN); 68° C. for 1:00

3) 40 cycles of

-   -   94° C. for 0:15; 52° C. for 0:20; 68° C. for 1:00

4) 68° C. for 3:00

5) 4° C. hold

The reaction mixtures were used as template DNA for Step II (see below)without any further purification.

TABLE 8 Step I Primers for Amplifying Kappa Light ChainsForward Primer Pool SEQ ID NO: 5′ LVκ1/2 ATGAGGSTCCCYGCTCAGCTGCTGG 87 5′LVκ3 CTCTTCCTCCTGCTACTCTGGCTCCCAG 88 5′ LVκ4 ATTTCTCTGTTGCTCTGGATCTCTG89 Reverse Primer SEQ ID NO: VK-Rev GCACTCTCCCCTGTTGAAGCTCTTTG 90

TABLE 9 Step I Primers for Amplifying IgG Heavy ChainsForward Primer Pool SEQ ID NO: 5′ L-VH1 ACAGGTGCCCACTCCCAGGTGCAG 91 5′L-VH3 AAGGTGTCCAGTGTGARGTGCAG 92 5′ L-VH4/6 CCCAGATGGGTCCTGTCCCAGGTGCAG93 5′ L-VH5 CAAGGAGTCTGTTCCGAGGTGCAG 94 Reverse Primer SEQ ID NO: 3′CγCH1 GGAAGGTGTGCACGCCGCTGGTC 95

Step II. Amplification

In Step II, the reaction mixtures from Step I were used as templates forsecond PCR reactions with pools of forward and reverse primers foreither the light chain or heavy chain, respectively. These reactionsamplified the DNA from the framework 1 region of each chain. The lightchain forward primers (see Table 10) were designed to introduce a SfiIrestriction site (SEQ ID NO:41). The reaction conditions were asfollows:

PCR II: Light Chain

H₂O 15.75 10x buffer 2.5 10X Enhancer 2.5 dNTP (10 mM each) 0.75 Step Ireaction 2.5 Vk Primer Pool (9.1 μM) 0.5 pCALCK(G)L (20 μM) 0.25 Pfx500.25 25 μL

The heavy chain forward primers (see Table 11) were designed tointroduce a SalI restriction site (SEQ ID NO:96). The reactionconditions were as follows:

PCR II: Heavy Chain

H₂O 14.25 10x buffer 2.5 10X Enhancer 2.5 dNTP (10 mM each) 0.75 Step Ireaction 2.5 pCAL24VH-F pool (2 μM) 2 SalI JH-Rev pool (20 μM) 0.25Pfx50 0.25 25 μL

The PCR thermocycler conditions were as follows:

1) 94° C. for 2 minutes

2) 50 cycles of:

-   -   94° C. for 15 seconds; 54° C. for 20 seconds; 68° C. for 1        minute

3) 68° C. for 3 minutes

4) 4° C. hold

Following amplification, the PCR reaction products were separated on a1% agarose gel and the band corresponding to the heavy chain (400 bp)and the light chain (650 bp) were purified by gel extraction (Qiagen).

TABLE 10 Primers for Amplifying Kappa Light Chains Forward Primer PoolSEQ ID NO: VK1a AAggcccagccggccatggccgccggtGACATCCAGATGACCCAG 57 VK1bAAggcccagccggccatggccgccggtGACATCCAGTTGACCCAG 58 VK1cAAggcccagccggccatggccgccggtGCCATCCGGTTGACCCAG 59 VK2aAAggcccagccggccatggccgccggtGATATTGTGATGACYCAG 60 VK3aAAggcccagccggccatggccgccggtGAAATTGTGTTGACGCAG 61 VK3bAAggcccagccggccatggccgccggtGAAATTGTGTTGACACAG 62 VK3cAAggcccagccggccatggccgccggtGAAATAGTGATGACGCAG 63 VK4aAAggcccagccggccatggccgccggtGACATCGTGATGACCCAG 64 VK5aAAggcccagccggccatggccgccggtGAAACGACACTCACGCAG 65 VK6aAAggcccagccggccatggccgccggtGAAATTGTGCTGACTCAG 66 VK6bAAggcccagccggccatggccgccggtGATGTTGTGATGACACAG 67 Reverse PrimerSEQ ID NO: pCALCK (G) L CTCCTTATTAATTAATTAGCACTCTCCCCTGTTGAAGCTCTTTG 68

TABLE 11 Primers for Amplifying IgG Heavy Chains Forward Primer PoolSEQ ID NO: pCal30 VH1a ggctttgctaccgtagcgCAGGCGGCCGCACAGGTKCAGCTGGTGCAG42 pCal30 VH1b ggctttgctaccgtagcgCAGGCGGCCGCACAGGTCCAGCTTGTGCAG 43pCal30 VH1c ggctttgctaccgtagcgCAGGCGGCCGCASAGGTCCAGCTGGTACAG 44pCal30 VH1d ggctttgctaccgtagcgCAGGCGGCCGCACARATGCAGCTGGTGCAG 45pCal30 VH2a ggctttgctaccgtagcgCAGGCGGCCGCACAGATCACCTTGAAGGAG 46pCal30 VH3a ggctttgctaccgtagcgCAGGCGGCCGCAGARGTGCAGCTGGTGGAG 47pCal30 VH4a ggctttgctaccgtagcgCAGGCGGCCGCACAGSTGCAGCTGCAGGAG 48pCal30 VH4b ggctttgctaccgtagcgCAGGCGGCCGCACAGGTGCAGCTACAGCAG 49pCal30 VH5a ggctttgctaccgtagcgCAGGCGGCCGCAGARGTGCAGCTGGTGCAG 50pCal30 VH6 ggctttgctaccgtagcgCAGGCGGCCGCACAGGTACAGCTGCAGCAG 51pCal30 VH7 ggctttgctaccgtagcgCAGGCGGCCGCACAGGTSCAGCTGGTGCAA 52Reverse Primer Pool SEQ ID NO: 3′ SalIJH 1/2/4/5TGCGAAGTCGACGCTGAGGAGACGGTGACCAG 97 3′ SalIJH3TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG 98 3′ SalIJH6TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG 99

Step III. Overlap PCR

In Step III, the heavy chain and light chain DNA segments generated instep II were: 1) linked in an overlap reaction with a Fab linker (seeTable 12, below) that anneals to the 3′ end of the light chain and the5′ end of the heavy chain and 2) amplified with a Sfi forward primer(see Table 12, below) that anneals to the 5′ end of the light chain andJH reverse primers (see Table 11, above) that anneal to the 3′ end ofthe heavy chain, thereby allowing amplification of a 1200 base pair (bp)antibody fragment containing the light chain-linker-heavy chain. Thereaction conditions were as follows (the linker was generated asdescribed in Example 2 above):

H₂O 24.5 10x buffer 5 10X Enhancer 5 dNTP (10 mM each) 1.5 Light Chain 5Heavy Chain 5 Linker 2 Sfi F/JH-R Primers (20 μM) 1 Pfx50 1 50 μL

The PCR thermocycler conditions were as follows:

-   -   Overlap with Linker        -   1) 94° C. for 2 minutes        -   2) 15 cycles of:            -   94° C. for 15 seconds: 68° C. for 1 minute    -   Add primers        -   3) 94° C. for 2 minutes        -   4) 30 cycles of:            -   94° C. for 15 seconds; 60° C. for 20 seconds; 68° C. for                1 minute        -   5) 68° C. for 3 minutes        -   6) 4° C. hold

Following amplification, the PCR reaction product lightchain-linker-heavy chain was separated on a 1% agarose gel and waspurified by gel extraction (Qiagen).

Step IV. Introduction of C_(H)1 region

Following overlap, the amplified light chain-linker-heavy chain wasdigested with Sal I and ligated to a Sal1 digested heavy chain constantregion 1 (CHγ1 region) introducing a SfiI restriction site at the 3′ endof the heavy chain constant region. The ligation reaction conditionswere as follows:

2 μl of Ligation reaction buffer

2 μl of C_(H)1

5 μl of 1.2 kB gel purified product from step III

10 μl of water

1 μl T4 Ligase

The ligation reaction mixture was incubated for 30 minutes at roomtemperature.

Following ligation, the full length Fab was amplified by PCR with SfiIForward and Reverse primers (see Table 12, below) resulting in a 1.45 kbfragment. The reaction conditions were as follows:

H₂O 31.5 10x buffer 5 10X Enhancer 5 dNTP (10 mM each) 1.5 Ligationreaction mixture 5 Sfi F/R Primers (20 μM) 1 Pfx50 1 50 μL

The PCR thermocycler conditions were as follows:

-   -   1) 94° C. for 2 minutes    -   2) 30 cycles of:        -   94° C. for 15 seconds; 60° C. for 20 seconds; 68° C. for 1            minute    -   3) 68° C. for 3 minutes    -   4) 4° C. hold

The reaction product was a 1.45 kB fragment of a light chain and heavychain linked together in a single cassette.

TABLE 12 Step III and Step IV Oligonucleotides OligonucleotideSEQ ID NO: Fab Linker GAGCTTCAACAGGGGAGAGTGCTAATTAATTAATAAGGAGGat 100ataattatgaaaaagacagctatcgcgattgcaGTGGCACTGGCTGGCTTTGCTACCGTAGCGCAGGCGGCCGCA Sfi Forward TCGCggcccagccggccatggc 84Sfi Reverse TGCGGCCGGCCTGGCCGA 85 CH1 fragmentgtcgaccaaaggtccgtctgttttcccgctggctccgtcttct 101aaatctacctctggtggtaccgctgctctgggttgcctggttaaagactacttcccggaaccggttaccgtttcttggaactctggtgctctgacctctggtgttcacaccttcccggctgttctgcagtcttctggtctgtactctctgtcttctgttgttaccgttccgtcttcttctctgggtacccagacctacatctgcaacgttaaccacaaaccgtctaacaccaaagttgacaagaaagttgaaccgaaatcttgcctgcgatcgcggccaggccggccgcaccatcaccatcaccatggcgcatacccgtacgacgttccggactacgcttctac tagt

Step V. Digestion with Sfi and Cloning into pCAL Expression Vector

Following overlap PCR reaction and purification of the PCR product, thereaction product was digested with SfiI. To the 30 μl eluate (seeabove), the following was added for the digestion:

 4 μl Reaction buffer 2 (New England Biolabs) 0.4 μl BSA 1.6 μl SfiIenzyme (New England Biolabs)  4 μl H₂O  40 μl Total Volume

The reaction is incubated for 1 hour at 37° C. Following digestion, thedigested overlap product was separated on a 1% agarose gel and the bandcorresponding to the antibody (˜1.45 kB) was purified by gel extraction(Qiagen Gel Extraction Purification Kit Cat. No. 28706). Briefly, thegel slice was digested with 500 μl of buffer QC (Qiagen). 150 μl ofisopropanol was added to digest and the sample was applied to theQiaSpin column. The column was washed twice with buffer PE (Qiagen) andthe sample is eluted in 30 μl of EB buffer (Qiagen). About 15 ng/μl ofdigested sample is recovered from approximately 1 μg of PCR overlapproduct.

Finally, the digested overlap product was ligated into a pCAL bacterialexpression vector (SEQ ID NO:86). The ligation reaction conditions wereas follows:

25 ng SfiI digested pCAL vector 25 ng digested overlap product 1 μl T4Ligase (NEB Cat. No. MC202L, 400,000 Units/ml) 20 μl total volume

The sample was ligated for 1 hour at room temperature. 1 μl of theligation was diluted in 4 μl of H₂O before proceeding to transformation.

Step VI. Transformation into E. coli

Following ligation, the ligation product was transformed into DH5a MaxEfficiency cells (Invitrogen; Cat No. 18258; Genotype: F− φ80lacZΔM15Δ(lacZY A-argF) U169 recA1 endA1 hsdR17 (rk−, mk+) phoA supE44 λ− thi-1gyrA96 relA1). In short, 1 μl ligation product (1/5 dilution) was addedto 50 μl DH5α and incubated on ice for 30 minutes. Transformation waseffected by heat shock at 42° C. for 45 seconds followed by 2 minutes onice. 0.9 mL SOC medium was added and the cells were allowed to recoverat 37° C. for 1 hour with shaking. Cells were plated on LB platessupplemented with carbenicillin (100 μg/mL) and 20 mM glucose. Theplates were incubated overnight at 37° C.

Step VII. Selection of Individual Colonies.

A total of 88 individual colonies were selected and grown in 1 mL SuperBroth (SB) supplemented with 1 carbenicillin (100 μg/mL) in a 96-wellplate for 2 hours at 37° C. A daughter plate was generated bytransferring 500 μl of each culture into another 96-well formatbacterial plate with 500 μl of SB supplemented with 40 mM glucose (final20 mM) and 100 μg/ml of carbenicillin. The original or mother plate wasfed 500 μl of SB supplemented with 100 μg/ml carbenicillin. The originalplate was grown at 30° C. overnight and the daughter plate (containingglucose) was grown at 37° C. overnight. The cell lysate from the 30° C.plate was used for bacterial ELISAs (see Example 4 below) and the 37° C.plate cultures were used for mini-prep DNA preparations (Qiagen).

Example 4: Antibody Binding to RSV F Protein

In this example, Fab antibodies generated in Examples 2 and 3 weretested for their ability to bind to purified RSV F1 lysate by ELISA.Briefly, 50 μL bacterial cell lysate diluted 1 volume into 3 volumestotal with PBS/3% BSA/0.01% TWEEN®20 was added to a 96-well ELISA platepreviously coated with RSV F1 lysate (see Example 1, above). The platewas incubated at 37° C. for 2 hours, or alternatively at 4° C.overnight, followed by washing 4× with wash buffer (PBS/0.05% TWEEN®20).50 μL goat anti-human IgG F(ab)-HRP antibody (Jackson Labs Cat. No.109-036-097) diluted 1:1000 in PBS/3% BSA/0.01% TWEEN®20 was added andthe plate was incubated at 37° C. for 1 hour. Following washing 6× withwash buffer, 50 μL 1:1 v/v TMB:peroxide solution (Pierce, Cat No. 34021)was added and allowed to develop for 7 minutes. The reaction wasimmediately halted by the addition of 50 μL 2N H₂SO₄ and the absorbanceat 450 nm was measured using an ELISA plate reader. Positive binding wasindicated by an OD₄₅₀ greater than 0.5 (0.5-0.9 is moderate binding, >1is strong binding) and a response that was 3-fold above background.

In addition to binding to RSV F1 lysate, several positive and negativecontrol antigens were also utilized. Plasma from a pool of Blood Bankdonors (collected and frozen after Ficoll Hypaque separation, diluted1:1000) was used as a positive control for RSV F1 lysate binding. As apositive control to determine that each bacterial cell lysate containsan intact Fab, an Affinipure goat anti-human F(ab)2 antibody (1 μg/mlJackson Immunoresearch Cat. No. 109-006-097) was used to coat a 96-wellELISA plate to capture intact Fab. This antibody binds only to the F(ab)portion of an IgG antibody. Fab expression was then detected by usinganti-HA Peroxidase (Roche. Cat. #12013819001; the bacterial expressedFabs have an HA-tag). Actin (1 μg/ml, Sigma Cat. No. A3653) was used asa negative control for Fab binding to any protein and as a positivecontrol for the ELISA reaction using mouse anti-actin antibody (1.25μg/ml. Sigma Cat. No. A3853) and goat anti-mouse IgG F(ab)-HRP antibody(Santa Cruz Biotech Cat. No. SC3697). The mouse anti-RSV mAb (clone 2F7,mouse ascites fluid, Cat. No. ab43812. Abcam) was also included asnegative control for specificity of binding to the RSV F protein sincethis antibody was employed to bind RSV F protein to the ELISA plate andthus was present on the ELISA plates during screening of the humananti-RSV antibodies.

A. Binding of Cell Lysates for Fabs Generated from EBV-Transformed BCells (See Example 2, Sections a and C, Kappa Light Chains, Cloned intopCAL Vector)

Eighty-eight (88) cell lysates generated in Example 2 above were testedby ELISA for their ability to 1) bind to an anti-Fab antibody; and 2)bind RSV F1 lysate. ELISA confirmed that 76 of 88 cell lysates werepositive for Fab production while 59 of the 88 cell lysates bound RSV Flysate. Confirmation ELISA revealed that 72 of the 76 cell lysates wereindeed producing Fab and 46 of the initial 59 positive hits werereconfirmed as binders to RSV F lysate.

Three of the positive binders were identified by DNA sequencing of thecorresponding DNA prep. Sequencing revealed they all had the samesequence, identified as Fab 58c5, which has the following light andheavy chains:

Fab 58c5

Light Chain (SEQ ID NO: 5)EIVMTQSPSSLSASIGDPVTITCQASQDISTYLNWYQQKPGQAPRLLIYGASNLETGVPSPFTGSGYGTDFSVTISSLQPEDIATYYCQQYQYLPYTFAPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECHeavy Chain (SEQ ID NO: 1)QVQLVQSGPGLVKPSQTLALTCNVSGASINSDNYYWTWIRQRPGGGLEWIGHISYTGNTYYTPSLKSPLSMSLETSQSQFSLRLTSVTAADSAVYFCAACGAYVLISNCGWFDSWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

B. Binding of Cell Lysates for Fabs Generated from Single Cell Sorting(See Example 3)

The results indicated that 64 of 88 cell lysates generated in Example 3bound RSV F1 protein. Twenty four positive binders were identified byDNA sequencing of the corresponding DNA prep.

One of the positive binders identified was Fab sc5 which has thefollowing light and heavy chains:

Fab sc5

Light Chain (SEQ ID NO: 13)DIQMTQSPSSLSASVGDRVTITCRASQNIKNYLNWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYSCQQSYNNQLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECHeavy Chain (SEQ ID NO: 9)QVQLQESGPGLVKPSGTLSLTCTVSGDSISGSNWWNWVRQPPGKGLEWIGEIYYRGTTNYKSSLKGRVTMSVDTSKNQFSLKLTSVTAADTAVYYCARGGRSTFGPDYYYYMDVWGRGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

C. Binding of Cell Lysates for Fabs Generated from EBV-Transformed BCells (See Example 2, Sections B and C, Kappa or Lambda Light Chains,Cloned into pCAL IT* Vector)

For the following experiments, the ELISA assays were performed asdescribed above with the following modifications: Bacterial supernatantexpressing 58c5 and sc5 Fab were used as positive controls for bindingto RSV F1 lysate (recombinant source) and HEp2 lysate (native RSV Fsource), respectively, at a 1:3 dilution in PBS containing 3% BSA and0.01% TWEEN®20.

Fab 30D8

For 30D8, 77 cell lysates generated in Example 2 above were tested byELISA for their ability to 1) bind to an anti-Fab antibody; and 2) bindRSV F1 lysate. ELISA confirmed that 70 cell lysates were positive forFab production while 63 of the 77 cell lysates bound RSV F lysate.Confirmation ELISA on 8 clones revealed that 8 of the 8 cell lysateswere indeed producing Fab and 6 of the 8 hits tested were reconfirmed asbinders to RSV F lysate. Three of the positive binders were sequencedand all contained the same antibody with the following light and heavychains (30D8):

Light Chain (SEQ ID NO: 395)QSVLTQASSVSVAPGQTARITCGANNIGSQNVHWYQQKPGQAPVLVVYDDRDRPSGIPDRFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSRDQAVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTIAPTECSHeavy Chain (SEQ ID NO: 396)EVQLLQSGAELKKPGASVKISCKTSGFTFSGHTIAWVRQAPGQGLEWMGWVSTNNGNTEYAQKIQGRVTMTMDTSTSTVYMELRSLTSDDTAVYFCAREWLVMGGFAFDHWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

Fab 104E5

For 104E5, 88 cell lysates generated in Example 2 above were tested byELISA for their ability to 1) bind to an anti-Fab antibody; and 2) bindRSV F1 lysate. ELISA confirmed that 15 cell lysates were positive forFab production while 4 of the 88 cell lysates bound RSV F lysate.Confirmation ELISA on 24 clones revealed that 10 of the 24 cell lysateswere indeed producing Fab and 7 of the 24 hits tested were reconfirmedas binders to RSV F lysate. Two of the positive binders were sequencedand contained the same antibody with the following light and heavychains (104E5):

Light Chain (SEQ ID NO: 397)DIQMTQSPSSLPASVGDPVTITCRASQNIKTYLNWYQQKPGRAPKLLISAVSNLQSGVPSRFSGTGSGTDFTLTISSLQPEDFATYYCQQSFSIPLTEGGGAKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQG LSSPVTKSFNRGECHeavy Chain (SEQ ID NO: 398)QVQLEQSGAEVKKPGSSVKVSCKPSGGTFDTYTISWVRQAPGQRLEWLGRIIPSLGETNYAHKLQGRVTITADKATSVVYMDLSDLTSEDAAVYYCAERITGPVDWVWDYGMDVWGQGTTVSVSSASSKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

Fab 38F10

For 38F10, 88 cell lysates generated in Example 2 above were tested byELISA for their ability to 1) bind to an anti-Fab antibody; and 2) bindRSV F1 lysate. ELISA confirmed that 65 cell lysates were positive forFab production while 40 of the 88 cell lysates bound RSV F lysate.Confirmation ELISA on 16 clones revealed that 13 of the 16 cell lysateswere indeed producing Fab and 13 of the 16 positive hits tested werereconfirmed as binders to RSV F lysate. Five of the positive binderswere sequenced and all contained the same antibody with the followinglight and heavy chains (38F10):

Light Chain (SEQ ID NO: 399)DIQLTQSPPTLSASVGDRVSMTCRASQSISNWLAWYQQKPGKAPKLLIQKASNLEDGVPSRFTASGFGTEFTLTISSLQPDDFATYYCQQYNSYSGLSFGGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECHeavy Chain (SEQ ID NO: 400)EVQLLESGGDVVQPGKSLRLSCTASGESITDFGIHWVRQAPGKGLEWVALISYNEVNIHYGESVRGRFTISRDIAKNTVYLQMNGLRPEDTGVYFCARDVWEDSWLSLACFQEWGQGSLVVVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

Fab 14G3

For 14G3, 88 cell lysates generated in Example 2 above were tested byELISA for their ability to 1) bind to an anti-Fab antibody; and 2) bindRSV F1 lysate. ELISA confirmed that 71 cell lysates were positive forFab production while 13 of the 88 cell lysates bound RSV F lysate.Confirmation ELISA on 16 clones revealed that 14 of the 16 cell lysateswere indeed producing Fab and 5 of the 16 positive hits tested werereconfirmed as binders to RSV F lysate. Four of the positive binderswere sequenced and all contained the same antibody with the followinglight and heavy chains (14G3):

Light Chain (SEQ ID NO: 401)DVVMTQTPLSLSVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCMQRMEFPFTFGQGTRLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGECHeavy Chain (SEQ ID NO: 402)QVQLQESGPGLVKPSQTLSLTCTVSGASISSDNHYWSWIRQPPGKGLEWIASIYYTGGTNYNPSLKSRLALSIDTSGDQFSLKLSSVTAADTAVYYCVRGLFFITARPYWYFDLWGRGTLVAVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

Fab 90D3

For 90D3, 88 cell lysates generated in Example 2 above were tested byELISA for their ability to 1) bind to an anti-Fab antibody; and 2) bindRSV F1 lysate. ELISA confirmed that 80 cell lysates were positive forFab production while 2 of the 88 cell lysates hound RSV F lysate.Confirmation ELISA on 8 clones revealed that 7 of the 8 cell lysateswere indeed producing Fab and 3 of the 8 positive hits tested werereconfirmed as binders to RSV F lysate. Two of the positive binders weresequenced and each contained the same antibody with the following lightand heavy chains (90D3):

Light Chain (SEQ ID NO: 403)AIRLTQSPSSLSASVGDRVSITCRASQSISNFLNWYQQKPGRAPKLLISAASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTYISLYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECHeavy Chain (SEQ ID NO: 404)QVQLVESGGGLVKPGGSLRLSCVGSGFTLKNYEMNWVRQAPGQGLQYISYISSSGNVVKYVDSVQGRFTISRDNAGNSLYLQMNNLRAEDTATYYCVRGFSIDKYDSSVDEYWGQGIAVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

D. Additional Anti-RSV Antibodies

Three additional antibodies, Fabs 56E11, 17C9 and 69F6, were isolated asdescribed in Example 2, Sections B and C above, amplified using primersfor both kappa or lambda light chains. The sequences of the heavy andlight chains are set forth below.

Fab 56E11

Light Chain (SEQ ID NO: 453)QAVLTQPPSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPRLIISEVTKRPSGVPGRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGSRHVVEGGGTKLTVLGQPKAAPSVTLEPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTIAPAECSHeavy Chain (SEQ ID NO: 452)EVQLQESGPGLVKPSETLSLTCSVSGVSINSNNYFWAWIRQPPGKGLEWIGNIYYGGSTHYNASLQSRVTISVDTSKSQFSLKLNSVTSADTAVYYCAASESIFWDYYYGLDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

Fab 17C9

Light Chain (SEQ ID NO: 455)EIVLTQSPSTLSASVGDRVTITCRASQNINTWLAWYQQKPGKAPKLLIYAASFLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYFCQQANSFPRTFGQGTKVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECHeavy Chain (SEQ ID NO: 454)QVQLQQWGAGLVRPSETLSLTCAVYGDSENDYFWTWIRQTPGKGLEWIGEISHSGSTNYSPSLKSRVTISVDTSKNQFSLKLSAVTAADTTVYFCARGVRSRPPPSYRGSGSPPYYHYGMDVWGQGITVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC

Fab 69F6

Light Chain (SEQ ID NO: 457)EIVLTQSPSSLSASVGDRVTISCQASQDISNYLNWYQQKPGKAPRLLIYDASYLDTGVPSRFSGSGSGTDETETISSLQPEDFATYYCQQYDDLRGGFTEGPGTKVDVKRTVAAPSVFIFRPSDEQLKSGTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECHeavy Chain (SEQ ID NO: 456)QMQLVQSGAEVRKPGESLKIACKGSGYSFTSYWIAWVRQMPGKGLEWLGIIFPNDSDATYSPSFQGQVTMSVDKSISTAYLQWNSLKASDTAVYFCARQYYLGSFESWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSC

The antibody domains and CDR regions of the isolated Fabs are providedin Tables 13A-13C below.

TABLE 13A Antibody domains and VH CDR regions of isolated Fabs Heavy Abchain VH domain VH CDR1 VH CDR2 VH CDR3 58c5 SEQ ID Amino acidsGASINSDNYYWT HISYTGNTYYTPSLKS CGAYVLISNCGWFDS NO: 1 1-125 of SEQ(SEQ ID NO: 2) (SEQ ID NO: 3) (SEQ ID NO: 4) ID NO: 1 sc5 SEQ IDAmino acids GDSISGSNWWN EIYYRGTTNYKSSLKG GGRSTFGPDYYYYMDV NO: 91-125 of SEQ (SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12) ID NO: 930D8 SEQ ID Amino acids GFTFSGHTIA WVSTNNGNTEYAQKIQG EWLVMGGFAFDHNO: 396 1-121 of SEQ (SEQ ID NO: 405) (SEQ ID NO: 406) (SEQ ID NO: 407)ID NO: 396 104E5 SEQ ID Amino acids GGTFDTYTIS RIIPSLGETNYAHKLQGRITGPVDWVWDYGMDV NO: 398 1-125 of SEQ (SEQ ID NO: 411) (SEQ ID NO: 412)(SEQ ID NO: 413) ID NO: 398 38F10 SEQ ID Amino acids GFSITDFGIHLISYNEVNIHYGESVRG DVWEDSWLSLACFQE NO: 400 1-124 of SEQ (SEQ ID NO: 417)(SEQ ID NO: 418) (SEQ ID NO: 419) ID NO: 400 14G3 SEQ ID Amino acidsGASISSDNHYWS SIYYTGGTNYNPSLKS GLFFITARPYWYFDL NO: 402 1-125 of SEQ(SEQ ID NO: 423) (SEQ ID NO: 424) (SEQ ID NO: 425) ID NO: 402 90D3SEQ ID Amino acids GFTLKNYEMN YISSSGNVVKYVDSVQG GFSIDKYDSSVDEY NO: 4041-123 of SEQ (SEQ ID NO: 429) (SEQ ID NO: 430) (SEQ ID NO: 431)ID NO: 404 56E11 SEQ ID Amino acids GVSINSNNYFWA NIYYGGSTHYNASLQSSESIFWDYYYGLDV NO: 452 1-124 of SEQ (SEQ ID NO: 458) (SEQ ID NO: 459)(SEQ ID NO: 460) ID NO: 452 17C9 SEQ ID Amino acids GDSFNDYFWTEISHSGSTNYSPSLKS GVRSRPPPSYRGSGSPPY NO: 454 1-133 of SEQ(SEQ ID NO: 464) (SEQ ID NO: 465) YHYGMDV ID NO: 454 (SEQ ID NO: 466)69F6 SEQ ID Amino acids GYSFTSYWIA IIFPNDSDATYSPSFQG QYYLGSFES NO: 4561-118 of SEQ (SEQ ID NO: 470) (SEQ ID NO: 471) (SEQ ID NO: 472)ID NO: 456

TABLE 13B Antibody domains and VL CDR regions of isolated Fabs Light Abchain VL domain VL CDR1 VL CDR2 VL CDR3 58c5 SEQ ID Amino acidsQASQDISTYLN GASNLET QQYQYLPYT NO: 5 1-107 of SEQ (SEQ ID NO: 6)(SEQ ID NO: 7) (SEQ ID NO: 8) ID NO: 5 sc5 SEQ ID Amino acidsRASQNIKNYLN AASTLQS QQSYNNQLT NO: 13 1-107 of SEQ (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 16) ID NO: 13 30D8 SEQ ID Amino acidsGANNIGSQNVH DDRDRPS QVWDSSRDQAVI NO: 395 1-110 of SEQ (SEQ ID NO: 408)(SEQ ID NO: 409) (SEQ ID NO: 410) ID NO: 395 104E5 SEQ ID Amino acidsRASQNIKTYLN AVSNLQS QQSFSIPLT NO: 397 1-107 of SEQ (SEQ ID NO: 414)(SEQ ID NO: 415) (SEQ ID NO: 416) ID NO: 397 38F10 SEQ ID Amino acidsRASQSISNWLA KASNLED QQYNSYSGLS NO: 399 1-108 of SEQ (SEQ ID N0: 420)(SEQ ID NO: 421) (SEQ ID NO: 422) ID NO: 399 14G3 SEQ ID Amino acidsRSSQSLLDSDDGNTYLD TLSYRAS MQRMEFPFT NO: 401 1-113 of SEQ(SEQ ID NO: 426) (SEQ ID NO: 427) (SEQ ID NO: 428) ID NO: 401 90D3Amino acids SEQ ID RASQSISNFLN AASSLQG QQTYISLYT NO: 403 1-107 of SEQ(SEQ ID NO: 432) (SEQ ID NO: 433) (SEQ ID NO: 434) ID NO: 403 56E11SEQ ID Amino acids TGTSSDVGGYNYVS EVTKRPS SSYAGSRHVV NO: 4531-111 of SEQ (SEQ ID NO: 461) (SEQ ID NO: 462) (SEQ ID NO: 463)ID NO: 453 17C9 SEQ ID Amino acids RASQNINTWLA AASFLQS QQANSFPRT NO: 4551-107 of SEQ (SEQ ID NO: 467) (SEQ ID NO: 468) (SEQ ID NO: 469)ID NO: 455 69F6 SEQ ID Amino acids QASDISNYLN DASYLDT QQYDDLRGGFTNO: 457 1-109 of SEQ (SEQ ID NO: 473) (SEQ ID NO: 474) (SEQ ID NO: 475)ID NO: 457

TABLE 13C Heavy chain CDR1 (Kabat numbering) Ab VH CDR1 Ab VH CDR1 58C5SDNYYWT (SEQ ID NO: 435) 14G3 SDNHYWS (SEQ ID NO: 440) sc5GSNWWN (SEQ ID NO: 436) 90D3 NYEMN (SEQ ID NO: 441) 30D8GHTIA (SEQ ID NO: 437) 56E11 SNNYFWA (SEQ ID NO: 482) 104E5TYTIS (SEQ ID NO: 438) 17C9 DYFWT (SEQ ID NO: 483) 38F10DFGIH (SEQ ID NO: 439) 69F6 SYWIA (SEQ ID NO: 484)

Example 5: Expression and Purification of Isolated Fabs

In this example, individual Fab antibodies that were determined to bindRSV F lysate by ELISA using cell lysate were subsequently expressed andpurified from the bacterial cells using column chromatography.

The DNA encoding each individual Fab antibody was transformed into Top10cells (Invitrogen) for expression. Each Fab antibody was grown in 2 L SBat 37° C. to an OD₆₀₀ of 0.8. Protein expression was induced by theaddition of 1 mM IPTG and allowed to occur overnight at 30° C. Followingexpression, the bacterial cultures were centrifuged and the cell pelletwas resuspended in 10 mL Phosphate Buffered Saline (PBS) With proteaseinhibitors (Complete Protease Inhibitor Cocktail, Santa Cruz Biotech,Cat. # sc-29131). Lysozyme (0.2 mg) was added to the resuspended cellsand the mixture was incubated at room temperature for 15 minutes. Thecells were lysed by two freeze/thaw cycles. In short, the resuspendedbacterial cells were frozen in an ethanol/dry ice bath followed bythawing in a 37° C. water bath. Once lysed, the bacterial lysate wascentrifuged at 18000 rpm and the supernatant was filtered and sterilizedby passing through a 0.4 micron filter.

Each individual Fab antibody was then purified by affinity columnchromatography. In short, the filtered supernatant was passed slowlyover an anti-Fab/Protein A column allowing the Fab protein to bind.Following washing with 50 mL PBS, the bound Fab was eluted with 9 mL of0.2 M glycine, pH 2.2 and collected in a conical tube containing 1 mL of2M Tris, thereby neutralizing the eluted protein. The eluted Fab wasthen dialyzed using a 10K MWCO dialysis cassette (Pierce) against 4 LPBS. The protein was stored at 4° C. overnight and subsequentlyconcentrated to a volume of 1 mL using a 10 kDa Amicon Ultra Filter(Millipore). Binding of each purified Fab antibody to RSV F lysate(recombinant source, Example 1A) and HEp2 lysate (native source, Example1B) was then reconfirmed by ELISA (see Example 4 above). Additionally,each purified Fab antibody was tested for its ability to neutralize RSVusing the assay described in Example 6.

Binding of Fabs 58c5 and Sc5 to RSV F Lysate and Purified RSV F Protein

The binding of antibodies 58C5 and sc5 to either captured RSV F proteinfrom transfected 293 cells (recombinant) or purified RSV F protein fromRSV A2 infected Hep2 cells (native) was measured by ELISA. The resultsindicate that Fab 58c5 and Fab sc5 bind to RSV F protein (recombinant)in a dose dependent manner but only sc5 was able to recognize thepurified F protein (native) (see Tables 14-15 below).

TABLE 14 Binding of Fab sc5 and 58c5 to captured RSV F lysate(recombinant) Fab [μg/ml] sc5 58c5 2 2.963 2.9165 0.4 2.827 2.9705 0.082.151 2.518 0.016 0.651 1.433 0.0032 0.3205 0.5905 0.00064 0.284 0.4150.000128 0.337 0.3785 0.0000256 0.22 0.2485

TABLE 15 Binding of Fab sc5 and 58c5 to purified RSV-F Protein (native)Fab [μg/ml] sc5 58c5 2 2.623 0.417 0.4 2.704 0.2665 0.08 2.744 0.15050.016 2.66 0.098 0.0032 1.7685 0.0805 0.00064 0.6035 0.087 0.0001280.2325 0.1065 0.0000256 0.1445 0.13

Binding of Fabs 30D8, 104E5, 38F10, 90D3 and 14G3 to RSV F Lysate andPurified RSV F Protein

The estimated EC50s for binding of Fabs 30D8, 104E5, 38F10, 90D3 and14G3 are set forth in Table 16A below, Fabs 30D8 and 14G3 both bind RSVF1 lysate (recombinant) at an EC50 of approximately 5 nM (3.8 nM and 8.2nM, respectively). Fabs 104E5 and 90D3 bind native RSV strain A2 Fprotein at an EC50 of approximately 80 pM with Fab 38F10 binding nativeRSV strain A2 F protein at an EC50 of 177 pM.

TABLE 16A Estimated EC50 for Fabs 30D8, 104E5, 38F10, 90D3 and 14G3Purified Fab EC50 Antigen Source 30D8 3.8 nM RSV F1 Lysate (recombinant)104E5 80 pM HEp2 Lysate (native) 38F10 177 pM HEp2 Lysate (native) 90D372 pM HEp2 Lysate (native) 14G3 8.2 nM RSV F1 Lysate (recombinant)

Binding of Fabs 56E11, 17C9 and 69F6 to Purified RSV F Protein

The estimated EC50s for binding of Fabs 56E11, 17C9 and 69F6 are setforth in Table 16B below. Fab 56E11 binds RSV F1 lysate (recombinant)with an EC50 of 545 pM. Fabs 17C9 and 69F6 bind RSV F1 lysate(recombinant) with an EC50 of 136 pM and 135 pM, respectively.

TABLE 16B Estimated EC50 for Fabs 30D8, 104E5, 38F10, 90D3 and 14G3Purified Fab EC50 Antigen Source 56E11 545 pM RSV F1 Lysate(recombinant) 17C9 136 pM RSV F1 Lysate (recombinant) 69F6 135 pM RSV F1Lysate (recombinant)

Example 6: RSV Neutralization Assay

In this example, the anti-RSV antibodies were analyzed for their abilityto bind to and neutralize RSV virus in solution as assessed by a plaquereduction assay. In this experiment, the RSV virus and the antibodieswere pre-incubated in the absence of target cells. The mixture was thenadded to the cells and virus infection was measured by a standard plaquereduction assay described herein. The anti-RSV antibodies were analyzedfor their ability to neutralize several strains of RSV virus, includingRSV A2 (ATCC Cat. No. VR-1540), RSV B-wash (ATCC Cat. No. VR-1580,strain 18537), and RSV B-1 (ATCC Cat. No. 1400).

Vero cells (ATCC, cat no: CCL-81; Manassas, Va.) were employed for hostcell infection. Vero cells were grown in DMEM (HyClone, cat no: SH30285.01) with 10% fetal bovine serum (FBS) (HyClone, cat no:SH30070.03), supplemented with 1% L-Glutamine (HyClone, cat no:SH30034.01) and 1% Penicillin-Streptomycin solution (HyClone, cat no:SV30010). The Vero cells were maintained in a 37° C. incubator with 5%CO2 and passaged twice per week.

On day 1 of the experiment. Vero cells were cultured in 24-well cellculture plates. The cells were plated at a density (approximately 1×10⁶cells per well) which allows formation of a cell monolayers (>90%confluence) by day 2. On day 2, each antibody was serially diluted inplain Eagle's minimal essential medium (EMEM, ATCC, cat no: 30-2003)(final antibody concentrations tested: 20 μg/ml, 4 μg/ml, 0.8 μg/ml,0.16 μg/ml, 0.032 μg/ml, and 0.006 μg/ml). The RSV virus was alsodiluted in plain EMEM to a concentration of 2×10³ pfu/ml (100 pfu/50ul). And, 110 μl of the diluted RSV virus was added to 110 μl of eachdiluted antibody solution and mixed by pipetting. For the virus controlsample, 110 μl of the diluted RSV virus was added to 110 μl plain EMEM.The antibody-virus or virus control mixtures were incubated at 37° C.for 2 hours. Following incubation, the culture media was decanted fromthe 24-well cell culture plates containing the Vero host cells and 100μl of the pre-incubated virus-antibody or virus control mixture was thentransferred to each well. Each test and control sample was prepared intriplicate. The cells were then incubated at 37° C. for one hour withmixing every 15 min.

Following the incubation period, the culture media containing thevirus-antibody or virus control mixture was aspirated and 1 ml ofoverlay medium was added to each well (overlay medium contained EMEM, 2%FBS, 1% L-glutamine, 0.75% methylcellulose). The 24-well cell cultureplates were then incubated at 37° C. (with 5% CO₂) for approximately 72hours. Cell plates were fixed with 10% formalin for 1 hour at roomtemperature, washed 10 times with ddH₂O and blocked with 5% non-fat drymilk (NFDM) in PBS with 0.05% TWEEN® 20) at 37° C. for one hour.

Following incubation, the blocking solution was decanted and 200 μL ofmouse anti-RSV antibody (ab10018, Abcam, 1:1000 dilution in 5% NFDM) wasadded to each well. The plates were incubated at 37° C. for 2 hrs,washed 10 times with ddH₂O and 200 μL of goat anti-mouse HRP-conjugatedIgG (Pierce, Cat. No. 31432, 1:1000 dilution in 5% NFDM) was added toeach well. The plates were incubated at 37° C. for 2 hrs. The plateswere washed 10 times with ddH₂O and 2004 of TrueBlue™ peroxidasesubstrate (KPL Cat. No. 50-78-02) was added to each well. The plateswere developed for 10 min at room temperature. The plates were washedtwice with ddH20 and dried on a paper towel and the number of blueplaques was counted. The ED50 (effective dilution for 50%neutralization) was calculated using Prism (GraphPad). The plaquereduction rate was calculated according to the following formula:Plaque Reduction Rate(percentile)=(1−average plaque number in eachantibody dilution/average plaque number in virus control wells)*100

The data is shown in Tables 17-19 below. Table 17 lists the ED₅₀ foreach Fab for the various RSV strains. Table 18 lists the plaque countsfor the various RSV strains and at the varying concentrations for Fab58c5. Table 19 lists the plaque reduction rate for Fab 58c5. Table 20lists the plaque reduction rates for Fabs 56E11, 17C9, 69F6 and 30D8 forRSV A2. The results indicate Fabs 58c5 and 30D8 are capable ofneutralizing all three strains of RSV while Fab sc5 neutralizes only RSVA2 and RSV B-1, albeit at much higher antibody concentrations. Based onthe data obtained in the neutralization assay and the molecular weightof the Fab fragment (approximately 50 kDa), the ED₅₀ of Fab 58c5 for invitro neutralization of RSV A2 is approximately 320 pM whereas the ED₅₀of Fab 30D8 for in vitro neutralization of RSV A2 is approximately3-times lower (107 pM). Fab 30D8 is more effective against RSV B-1 andRSV B/wash than Fab 58c5.

TABLE 17 Fab Neutralization ED₅₀ Data Antigen RSV A2 RSV B-1 RSV B/wash58c5 320 pM 840 pM 500 pM (0.016 μg/mL) (0.042 μg/mL) (0.025 μg/mL) sc50.016 μM 0.042 μM >0.2 μM (0.8 μg/mL) (2.1 μg/mL) (>10 μg/mL) 30D8 107pM 51 pM 112 pM (5.35 ng/mL) (2.55 ng/mL) (5.6 ng/mL) 104E5 580 pM ND ND(29 ng/mL) 38F10 800 pM ND ND (40 ng/mL) 14G3 3400 pM ND ND (172 ng/mL)90D3 800 pM 560 pM 660 pM (40 ng/mL) (28 mg/mL) (33 ng/mL) 56E11 930 pMND ND (46.5 ng/mL) 17C9 352 pM ND ND (17.6 ng/mL) 69F6 260 pM ND ND (13ng/mL) ND = Not Determined

TABLE 18 Average Plaque Count for Neutralization with Fab 58c5 0.4 0.080.016 0.003 Antigen 10 μg/ml 2 μg/ml μg/ml μg/ml μg/ml μg/ml 0 μg/ml RSVA2 0 0 0 5.7 28.7 52.3 57.7 RSV B/wash 1.3 0.7 0 5 16.3 23.3 26.3 RSVB-1 0.3 0 0 4.7 8.7 11.7 12.3

TABLE 19 Plaque reduction rate (%) for Neutralization with Fab 58c5 0.40.08 0.016 0.003 Antigen 10 μg/ml 2 μg/ml μg/ml μg/ml μg/ml μg/ml 0μg/ml RSV A2 100 100 100 90 50 9.4 0 RSV B/wash 95 97 100 81 38 11 0 RSVB-1 97.6 100 100 62 29 5 0

TABLE 20 Plaque reduction rate (%) for Neutralization with Fabs 56E11,17C9 and 69F6 for RSV A2 0.4 0.08 0.016 0.0032 0.00064 Antibody 10 μg/ml2 μg/ml μg/ml μg/ml μg/ml μg/ml μg/ml 56E11 100 100 98.5 65.98 20.8 0 —17C9 100 100 99.9 91.2 46.7 3.4 — 69F6 99.7 99.8 99.6 97.5 63.0 2.3 —30D8 100 100 100 96.1 60.7 31.5 0.8 *30D8 was used as a positive control

Example 7: Cloning and Expression of IgG

In this example, Fab antibodies that showed potential to neutralize RSVwere converted into IgGs by cloning into the pCALM mammalian expressionvector (SEQ ID NO:102). Primers specific to each antibody were generatedand the heavy and light chains of each Fab as originally cloned into thepCAL vector (see Example 2) were amplified by PCR. Light chainamplification resulted in a 650 bp fragment and heavy chainamplification resulted in a 400 bp fragment. Additionally, a linker wasgenerated that allowed overlap of the heavy chain and the light chain.The linker also included a standard heavy chain constant region. Overlapof the heavy and light chains resulted in a 2.1 kB cassette for eachantibody that had SfiI restriction sites (SEQ ID NO:41) at both ends.Each cassette was digested with SfiI and cloned into the pCALM vector.After confirmation of the correct sequence in bacteria, DNA formammalian transfections was isolated using a Maxi Prep Kit (Qiagen).

To express each IgG, each pCALM vector was used to infect about 200million 293F cells resulting in about 200 micrograms of IgG. The 293Fcells were transfected with 293fectin (Invitrogen, Cat. No. 51-0031) andallowed to produce IgG for 72 hours. After 72 hours post transfection,the cell media was harvested, centrifuged to remove the cells, andfilter sterilized through a 0.4 micron filter unit. Purification waseffected by column chromatography using a Protein A column. The filteredmedia, containing the expressed IgG, was passed twice through a ProteinA column. Following washing with 50 mL of PBS, IgG was eluted with 9 mLof 0.2 M glycine at pH 2.2 and collected in 2 M Tris to effectneutralization. The elute was dialyzed against 4 liters of PBS using a10 kDa dialysis cassette (Pierce). The sample was concentrated down to 1mL with a 10 kDa Amicon Ultra (Millipore).

Example 8: IgG Binding Assays

In this example, the IgG forms of 58c5, 30D8, 104E5, 38F10, 14G3, 56E11,17C9 and 69F6, generated in Example 7 above, and anti-RSV antibodyMotavizumab (Wu et al. (2007) J. Mol. Biol. 368(3):652-665) were testedfor their ability to bind to RSV F protein (recombinant) or RSV Fprotein (native) lysate by ELISA (see Example 4 above). The estimatedEC50s for binding (determined by titrating each IgG) are set forth inTable 21 below. The IgG form of 30D8 has the highest affinity with theIgG forms of 58c5, 104E, 38F10, 56E11, 17C9 and 69F6 having about10-fold less affinity. Additionally, the IgG form of 30D8 has anaffinity for RSV strain A2 F protein about 10-fold greater thanmotavizumab.

TABLE 21 IgG Binding to RSV F Protein IgG EC₅₀ Antigen (estimated)Antigen Source IgG form of 58c5 24 pM RSV F1 Lysate (recombinant) IgGform of 30D8 2.5 pM  RSV F1 Lysate (recombinant) IgG form of 104E5 14.5pM  HEp2 Lysate (native) IgG form of 38F10 35 pM HEp2 Lysate (native)IgG form of 14G3 251 pM  RSV F1 Lysate (recombinant) IgG form of 56E1173 pM RSV F1 Lysate (recombinant) IgG form of 17C9 59 pM HEp2 Lysate(native) IgG form of 69F6 63 pM RSV F1 Lysate (recombinant) Motavizumab27 pM HEp2 Lysate (native)

Example 9: IgG RSV Neutralization Assays

In this example the IgG forms of 58c5, 30D8, 104E5, 38F10, 14G3, 56E11,17C9 and 69F6, generated in Example 7 above, and anti-RSV antibodyMotavizumab (Wu et al. (2007) J. Mol. Biol. 368(3):652-665) were testedfor their ability to neutralize various strains of RSV. Additionally,the IgG form of 58c5 was analyzed for its ability to neutralize variousmonoclonal antibody resistant RSV escape mutants (MARMs). A MARM is amutant RSV strain that is no longer capable of being neutralized by theantibody that it was generated against. Therefore, the ability of 58c5to neutralize a specific MARM indicates that the binding epitope of 58c5is different from that of the antibody to which the MARM was generated.

A. Neutralization of RSV

The IgG forms of 58c5, 30D8, 104E5, 38F10, 14G3, 56E11, 17C9 and 69F6were tested for their ability to neutralize RSV (as described in Example6 above). The data is shown in Tables 22-24 below. Table 22 lists theED₅₀ (effective dilution for 50% neutralization) for each RSV strain.Table 23 lists the plaque counts for the various RSV strains and atvarying antibody concentrations of the IgG form of 58c5. Table 24 liststhe plaque reduction rate for the various RSV strains and at varyingantibody concentrations of IgG 58c5. The results indicate the IgG formsof 58c5, 30D8, 104E5, 38F10, 14G3, 90D3, 56E11 and 17C9 are capable ofneutralizing all 3 strains of RSV, albeit at varying concentrations. TheIgG form of 69F6 is selective for the RSV strain A2. Based on the dataobtained in the neutralization assay and the molecular weight of the IgGfragment (approximately 150 kDa), the ED₅₀ of the IgG form of 58c5 forin vitro neutralization of RSV A2 is approximately 133 pM. The ED₅₀s forthe IgG forms of 30D8 and 14G3 for in vitro neutralization of RSV A2 are89 pM and 106 pM, respectively.

Comparison of the neutralization activity of the IgG form of 30D8 andanti-RSV antibody motavizumab (Wu et al (2007) J. Mol. Biol.368(3):652-665) for the three strains of RSV indicates IgG 30D8 is amore potent neutralizing antibody. For RSV strain A2, the IgG form of30D8 shows 4-fold improvement in neutralization (89 pM versus 360 pM).The IgG form of 30D8 is 15-fold better at neutralizing RSV strain B-1than motavizumab. For RSV strain B/wash, the IgG form of 30D8 shows a25-fold increase in neutralization (115 pM versus 2.9 nM). The IgG formof 30D8 was additionally shown to neutralize both the Long and B/9320strains of RSV.

TABLE 22 IgG Neutralization Data (ED₅₀) Antigen RSV A2 RSV B-1 RSVB/wash RSV Long RSV B/9320 IgG form of 58c5 133 pM 280 pM 193 pM ND ND(0.02 μg/mL) (0.042 μg/mL) (0.029 μg/mL) IgG form of 30D8 89 pM 55 pM115 pM 95 pM 85 pM (13.3 ng/mL) (8.3 ng/mL) (17.2 ng/mL) IgG form of104E5 800 pM 1.7 nM 8 nM ND ND (122 ng/mL) (253 ng/mL) (1.2 μg/mL) IgGform of 38F10 553 pM 1.4 nM 5.3 nM ND ND (83 ng/mL) (212 ng/mL) (789ng/mL) IgG form of 14G3 106 pM 10.7 nM 4.5 nM ND ND (16 ng/mL) (1.6μg/mL) (683 ng/mL) IgG form of 90D3 1.8 nM 1.4 nM 1.4 nM ND ND (263ng/mL) (211 ng/mL) (207 ng/mL) IgG form of 56E11 853 pM 295 pM 282 pM NDND IgG form of 17C9 363 pM 329 pM 860 pM ND ND IgG form of 69F6 225pM >67 nM >67 nM ND ND Motavizumab 360 pM 833 pM  2.9 nM ND ND

TABLE 23 Average Plaque Count for Neutralization with the IgG form of58c5 0.4 0.08 0.016 0.003 Antigen 10 μg/ml 2 μg/ml μg/ml μg/ml μg/mlμg/ml 0 μg/ml RSV A2 0.3 0 0.7 16.3 31 40.3 57.7 RSV B/wash 0 0 1.3 7.716.3 20.7 26.3 RSV B-1 0 0 0.3 4 9.3 11.7 12.3

TABLE 24 Plaque reduction rate (%) for Neutralization with the IgG formof 58c5 0.4 0.08 0.016 0.003 Antigen 10 μg/ml 2 μg/ml μg/ml μg/ml μg/mlμg/ml 0 μg/ml RSV A2 99.5 100 99 72 46 30 0 RSV B/wash 100 100 95 71 3821 0 RSV B-1 100 100 97.6 67.5 24 5 0

B. Neutralization of RSV Monoclonal Antibody Resistant RSV EscapeMutants

The IgG form of 58c5 was also tested for its ability to neutralizeseveral monoclonal antibody resistant RSV escape mutants (provided byDr. James Crowe, Vanderbilt University), as described in Example 6above. The MARMS, listed in Table 23 below, were derived from RSVwild-type strain A2. MARM 19, generated against human Fab 19 (see, e.g.,Crowe et al., Virology, 252:373-375 (1998)), contains the amino acidmutation isoleucine 266 to methionine. MARM 151, generated againstmurine mAb 151, contains the amino acid mutation lysine 272 toasparagine. MARM 1129, generated against the murine mAb 1129 which isthe parental antibody to palivizumab (SYNAGIS), contains the amino acidmutation serine 275 to phenylalanine.

The IgG form of 58c5 was also tested for its ability to neutralizeseveral RSV Monoclonal Antibody Resistant Mutants (MARMs). The data isshown in Tables 25-26 below. Table 25 lists the plaque counts forneutralization against the various MARMs at varying antibodyconcentrations. Table 26 lists the plaque reduction rate forneutralization against the various MARMs at varying antibodyconcentrations. The results indicate the IgG form of 58c5 is capable ofneutralizing all 3 RSV MARMS. Thus, the IgG form of 58c5 binds adifferent epitope of RSV strain A2 than Fab 19, murine mAb 1129 andmurine mAb 151.

TABLE 25 Average Plaque Count for Neutralization of IgG form of 58c5versus RSV MARMS 0.4 0.08 0.016 0.003 0 MARM 10 μg/ml 2 μg/ml μg/mlμg/ml μg/ml μg/ml μg/ml MARM 19 0.7 16.7 72 89.3 135 143 156 MARM 151 015.3 64.7 112 128 151 151 MARM 1129 0 0 2.3 5.7 11.7 17.7 22.3

TABLE 26 Plaque reduction rate for Neutralization of IgG form of 58c5versus RSV MARMS 0.4 0.08 0.016 0.003 0 MARM 10 μg/ml 2 μg/ml μg/mlμg/ml μg/ml μg/ml μg/ml MARM 19 100 89 53.8 42.7 14 8 0 MARM 151 100 9057 26 15 0 0 MARM 1129 100 100 90 74 47 21 0

Example 10: Competition Assays

In this example, competition assays were performed in which Fabs 58c5,30D8, 56E11, 17C9 and 69F6 were tested for their ability to competeagainst Motavizumab IgG (Wu et al. (2007) J. Mol. Biol. 368(3):652-665)for binding to the RSV F protein. Fabs 58c5 and 30D8 were additionallytested for their ability to compete against the IgG forms of 17C9 and69F6 for binding to recombinant RSV F protein. Fabs 56E11, 17C9 and 69F6were additionally tested for their ability to compete against the IgGforms of 58c5 and 30D8 for binding to recombinant RSV F protein. Aspositive controls for competition, the IgG forms of the variousantibodies were tested for their ability to compete against therespective Fab forms of the same antibodies.

Briefly, ELISA plates were prepared as described in Example 1 above,with either recombinant or native RSV strain A2 F protein. The plateswere blocked with 4% nonfat dry milk in 1×PBS for 2 hours at 37° C.followed by washing 4× with wash buffer (PBS/0.05% TWEEN®20). Fabs weretitrated in PBS/3% BSA/0.01% TWEEN®20 from 9 μg/mL to 0.0001 μg/mL(actual concentrations tested: 9, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003,0.001, 0.0003, 0.0001 μg/mL). The IgG forms of the tested antibodies andMotavizumab were added at fixed concentrations of either 0.5 μg/mL, 0.1μg/mL, 0.05 μg/mL and 0.01 μg/mL (as indicated in Table 27 below). 50 μLeach of diluted Fab and fixed concentration of IgG was addedsimultaneously to each well of a plate, in duplicate, as indicated inTable 27 below, and the plates were incubated at 37° C. for 2 hoursfollowed by washing 4× with wash buffer. Goat anti-human IgG Fc-gammaHRP (Jackson ImmunoResearch, Cat. No. 109-035-098, diluted 1:1000, wasadded and the plates were incubated at 37° C. for 1 hour. Followingwashing 6× with wash buffer, 50 μL 1:1 v/v TMB:peroxide solution(Pierce, Cat No. 34021) was added and allowed to develop for 7 minutes.The reaction was immediately halted by the addition of 50 μL 2N H₂SO₄and the absorbance at 450 nm was measured using an ELISA plate reader.

TABLE 27 Competition Assays Antigen Recombinant F protein Native Fprotein Fab 30D8 0.05 μg/mL 0.5 μg/mL (9 to 0.0001 μg/mL) IgG form of30D8 IgG form of 30D8 0.05 μg/mL 0.05 μg/mL IgG form of 58c5 IgG form of58c5 0.1 μg/mL 0.01 μg/mL motavizumab IgG motavizumab IgG Fab 58c5 0.5μg/mL 0.5 μg/mL (9 to 0.0001 μg/mL) IgG form of 30D8 IgG form of 30D80.05 μg/mL 0.05 μg/mL IgG form of 58c5 IgG form of 58c5 0.1 μg/mL 0.01μg/mL motavizumab IgG motavizumab IgG

The results are summarized in Table 28 below. Fabs 58C5, 30D3, 56E11,17C9 and 69F6 do not compete for binding to RSV strain A2 F protein withMotavizumab. Fab 30D8 did not compete for binding with any of the testedantibodies. Fab 58C5 competed with the IgG form of 69F6 for binding toRSV F protein. Fabs 56E11 and 69F6 both competed with the IgG form of58c5 for binding to the RSV F protein. Antibodies 58C5, 69F6 and 56E11share common epitope binding sites on the RSV fusion protein.

TABLE 28 Summary of Competition Assays 58C5 30D8 56E11 17C9 69F6 Fab FabFab Fab Fab Motavizumab IgG NO NO NO NO NO IgG form of 58C5 YES NO YESNO YES IgG form of 30D8 NO YES NO NO NO IgG form of 56E11 YES NO ND NDND IgG form of 17C9 NO NO ND YES ND IgG form of 69F6 YES NO ND ND YESND: Not Determined

Example 11: RSV MARM Generation and Neutralization Assays

In this example, monoclonal antibody resistant RSV escape mutants(MARMs) were generated for Motavizumab and the IgG form of 58C5. For theIgG form of 30D8, 12 rounds of selection were performed and no escapemutants were identified. Motavizumab, the IgG forms of 58C5 and 30D8 andFabs 56E11, 17C9 and 69F6 were further analyzed for their ability toneutralize the newly generated MARMs.

A. MARM Generation

1. Motavizumab

The concentration of motavizumab IgG that reduces RSV viral titers by 3logs (corresponding to 99.9% inhibition of RSV A2 virus byneutralization assay) was previously determined to be 3.2 μg/mL. RSV A2viral particles (2×10⁶) were preincubated with dilutions of motavizumabIgG and this mixture was used to infect Vero cell monolayers (asdescribed in Example 6 above). Wells with the highest antibodyconcentrations still demonstrating cytotoxic effects were selected foradditional rounds of selection. After 10 rounds of selection, plaquesfrom virus grown in the presence of 8 μg/mL motavizumab were obtained.Virus particles from these plaques were tested in neutralization assays(as described in Example 6 above) and RNA from positive particles wasprepared using a RNeasy extraction kit (Qiagen). Six escape mutants wereselected and the F gene was amplified by PCR. The DNA was sequenced andall six clones encoded a single amino acid substitution of glutamic acidfor lysine at position 272 (K272E, SEQ ID NO:486) compared to theparental RSV A2 strain (set forth in SEQ ID NO:485).

Table 29 below sets forth the highest antibody concentrationdemonstrating cytopathic effects (CPE) for each round of selection. Asshown in Table 29 below, motavizumab escape mutants were identifiedafter 7 rounds of selection, as identified by an antibody concentrationdemonstrating CPE greater than the concentration of motavizumab thatcorresponds to 99.9% inhibition of RSV A2 virus as determined byneutralization assay (i.e., >3.2 μg/mL).

TABLE 29 Motavizumab MARM Selection Selection Antibody ConcentrationRound (μg/mL) 1 0.5 2 0.5 3 0.75 4 1 5 2 6 3 7 4 8 8 9 8 10 8

2. 58C5

The concentration of the IgG form of 58C5 that reduces RSV viral titersby 3 logs (corresponding to 99.9% inhibition of RSV A2 virus byneutralization assay) was determined to be 0.8 μg/mL. RSV A2 viralparticles (2×10⁶) were preincubated with dilutions of the IgG form of58C5 and this mixture was used to infect Vero cell monolayers (asdescribed in Example 6 above). Wells with the highest antibodyconcentrations still demonstrating cytotoxic effects were selected foradditional rounds of selection. After 12 rounds of selection, plaquesfrom virus grown in the presence of 2 μg/mL of the IgG form of 58C5 wereobtained. Virus particles from these plaques were tested inneutralization assays (as described in Example 6 above) and RNA frompositive particles was prepared using a RNeasy extraction kit (Qiagen).Five escape mutants were selected and the F gene was amplified by PCR.The DNA was sequenced and all five clones encoded three amino acidsubstitutions (N63K, M115K and E295G; SEQ ID NO:487) compared to theparental RSV A2 strain (set forth in SEQ ID NO:485).

Table 30 below sets forth the highest antibody concentrationdemonstrating cytopathic effects (CPE) for each round of selection. Asshown in Table 30 below, 58C5 escape mutants were identified after 10rounds of selection, as identified by an antibody concentrationdemonstrating CPE greater than the concentration of the IgG form of 58C5that corresponds to 99.9% inhibition of RSV A2 virus as determined byneutralization assay (i.e., >0.8 μg/mL).

TABLE 30 58C5 MARM Selection Selection Antibody Concentration Round(μg/mL) 1 0.2 2 0.2 3 0.3 4 0.4 5 0.6 6 0.6 7 0.6 8 0.6 9 0.6 10 1.2 111.6 12 2

3. 30D8

The concentration of the IgG form of 30D8 that reduces RSV viral titersby 3 logs was determined to be 1 μg/mL. RSV A2 viral particles (2×10⁶)were preincubated with dilutions of the IgG form of 30D8 and thismixture was used to infect Vero cell monolayers (generated as describedin Example 6 above). Wells with the highest antibody concentrationsstill demonstrating cytotoxic effects were selected for additionalrounds of selection. After 12 rounds of selection, no 30D8 escapemutants were identified, as shown in Table 31 below.

TABLE 31 30D8 MARM Selection Selection Antibody Concentration Round(μg/mL) 1 0.2 2 0.2 3 0.3 4 0.4 5 0.4 6 0.4 7 0.4 8 0.4 9 0.4 10 0.4 110.4 12 0.4

B. Neutralization Assays

Motavizumab, the IgG forms of 58C5 and 30D8 and Fabs 56E11, 17C9 and69F6 were tested for their ability to neutralize the RSV A2 parentalvirus strain, the motavizumab MARM and the 58C5 MARM. The neutralizationassay procedure is described in Example 6 above. The data is shown inTables 32-35 below. Table 32 lists the plaque reduction rate forneutralization against the RSV A2 parental virus. Table 33 lists theplaque reduction rate for neutralization against the Motavizumab MARM.Table 34 lists the plaque reduction rate for neutralization against the58c5 MARM. Table 35 is a summary of the neutralization data (ED₅₀values).

The results indicate all antibodies are capable of neutralizing theparental RSV A2 strain, with the IgG forms of 58c5 and 30D8 showing thestrongest activity (see Tables 32 and 35). All of the tested antibodiesstrongly neutralize the Motavizumab MARM with no difference compared tothe parental strain (see Tables 33 and 35). As expected, motavizumabcannot neutralize the motavizumab MARM at any of the testedconcentrations. Motavizumab, the IgG form of 30D8 and Fab 17C9 stronglyneutralize the 58C5 MARM with no difference in neutralization potency(see Tables 34 and 35). As expected, the IgG form of 58c5 cannotneutralize the 58c5 MARM at any of the tested concentrations. Theresults show that the IgG forms of 58c5 and 30D8 and Fabs 56E11, 17C9and 69F6 all neutralized the motavizumab MARM indicating no competition.Thus, the epitopes of 58c5, 30D8, 56E11, 17C9 and 69F6 do not overlapwith that of motavizumab, as no competition was observed (see Table 28above) and all antibodies neutralized the motavizumab MARM.

Fab 69F6 competed with the IgG form of 58c5 for binding to the RSV Fprotein (see Table 28 above) but still neutralized the 58c5 MARM, albeitwith a slightly higher ED₅₀ than that for neutralization of the parentalA2 strain (353 pM versus 256 pM). In contrast, Fab 56E11 competed forbinding to the RSV F protein with the IgG form of 58c5 (see Table 28above) but did not neutralize the 58c5 MARM at any of the testedconcentrations. Thus, the 56E11 epitope overlaps with 58c5 as acompetition between the two antibodies is observed and 56E11 Fab wasunable to neutralize 58C5 MARM. Antibodies 69F6 and 17C9 neutralized the58c5 MARM indicating that their epitopes are not overlapping with thatof antibody 58c5. However, competition was observed between 58c5 and69F6 (see Table 28), but because 69F6 neutralized the 58c5 MARM, theobserved competition is more than likely due to steric hindrance.

TABLE 32 Plaque reduction rate for Neutralization of RSV A2 parentalvirus 10000 2000 400 80 16 Antibody ng/ml ng/ml ng/ml ng/ml ng/ml 3.2ng/ml Motavizumab 100.0 98.87 82.27 50.40 19.25 0.40 IgG form of 58C5100.0 100.0 99.6 87.0 49.2 6.1 IgG form of 30D8 100.0 100.0 96.2 81.058.2 11.9

TABLE 33 Plaque reduction rate for Neutralization of Motavizumab MARM10000 2000 400 80 Antibody ng/ml ng/ml ng/ml ng/ml 16 ng/ml 3.2 ng/mlMotavizumab 13.3 1.7 0.0 0.2 3.3 0.0 IgG form of 58C5 97.6 95.8 92.074.6 43.0 1.5 IgG form of 30D8 98.7 98.6 98.2 91.8 55.7 8.7

TABLE 34 Plaque reduction rate for Neutralization of 58C5 MARM 100002000 400 3.2 Antibody ng/ml ng/ml ng/ml 80 ng/ml 16 ng/ml ng/mlMotavizumab 94.8 92.9 83.9 47.1 6.5 0.0 IgG form of 58C5 3.3 0.0 0.0 0.00.0 0.0 IgG form of 30D8 93.6 87.7 83.9 80.0 58.1 8.4

TABLE 35 Summary of Neutralization (ED₅₀) values RSV A2 Motavizumab 58C5Parental MARM MARM (ED₅₀) (ED₅₀) (ED₅₀) Motavizumab IgG 519 pM >66.7 nM641 pM IgG form of 58C5 115 pM 173 pM >66.7 nM IgG form of 30D8 96 pM 93pM 113 pM 56E11 1 nM 1.6 nM >200 nM 17C9 436 pM 356 pM 353 pM 69F6 256pM 266 pM 353 pM

Example 12: Mapping of the 58C5 and 30D8 Binding Epitopes

In this example, residues of the RSV F protein involved in binding ofantibodies 58C5 and 30D8 were determined by screening a library ofsingle mutants of the RSV F protein for binding to antibodies 58C5 or30D8.

The RSV F protein mutant library \vas generated using ShotgunMutagenesis Mapping Technology from Integral Molecular (Philadelphia,Pa.). In short, the technology allows for the expression and analysis oflarge libraries of mutated target proteins within eukaryotic cells.Every residue in a target protein is individually mutated, usually tomultiple other amino acids, in order to assay for changes in monoclonalantibody (MAb) binding or function. The proteins are expressed withinmammalian cell lines, so even complex proteins that require eukaryotictranslational or post-translational processing can be epitope mapped.

Human RSV F A2 Strain (NCBI Ref # FJ614814) was used as the parentalplasmid for the RSV F protein. A C-terminal V5 tag was included to allowfor screening. The F proteins were expressed in HEK 293 cells andbinding was determined using immunoluminescence. Using a random mutationstrategy, a library of RSV F proteins containing 1029 mutant clones wasgenerated. All 549 amino acids of the F protein were mutated, with anaverage of 1.34 mutations per clones and an average of 2.46 mutationsper amino acid residue. Every amino acid was mutated at least once(100%), and at least 457 amino acids (83.2%) were mutated twice.Motivizumab and a polyclonal serum that binds the F protein were used aspositive controls to determine if the expressed F protein was properlyfolded. Motivizumab is known to bind both the pre- and the post-fusionconformation of the RSV F protein. In addition, both 58C5 and 30D8 aresensitive to the conformational changes, therefore, 58C5 was also usedas a control for 30D8 and 30D8 was used as a control for 58C5. As shownin Example 10 above, 58C5 and 30D8 bind to two independent sites of theRSV F protein and additionally, both are sensitive to conformationalchanges.

The screening was performed several times with two independent miniprepsof the transfectants. The selection criteria for the evaluation ofresidues that contribute to antibody binding was as follows:

-   -   mutant residue results in a signal for binding to 58C5 that is        2× less than the signal for binding to 30D8;    -   mutant residue results in a signal for binding to 30D8 that is        2× less than the signal for binding to 58C5;    -   positive control (polyclonal serum or Motivizumab)>20%        luminescence; and    -   preferably also another substitution at the same site.

Each identified clone was also verified for surface expression using apolyclonal antibody. Samples were tested at least 3 times.

1. Binding to Antibody 58C5

The results are shown in Tables 36-37 below, which set forth theimmunoluminescence for at least three samples (S1, S2 and S3) forbinding of 58C5 and 30D8 to each F protein mutant, binding ofmotavizumab and/or the polyclonal serum, the mean of the values for 58C5and 30D8, the ratio of binding to 30D8/58C5 and the mutation in the Fprotein. As shown in Tables 36-37 below, mutation of residues N63, E82,Y86, K168, V207, N371, S405 and/or E463 in the F protein resulted inreduced binding of the antibody 58C5. Residue E82 was identified in bothminiprep screens. Residue N63 was previously identified in the escapemutant, or MARK for 58C5. This mutation was also confirmed by FACSanalysis. When the combined identified residues are displayed on a 3Dmodel of prefusion RSV-F, they cluster on a restricted area of thespike, in the same area as the escape mutants in the top of the spikeand illustrate the antibody footprint of Mab 58C5 (see FIG. 1). Thus,58C5 binds a conformational epitope.

When these residues are displayed on the X-ray structure of thepostfusion RSV-F protein trimeric spike, the residues are distributedover a large area of the protein surface which would be impossible to bepart of an antibody footprint (see. FIG. 2, and Morton et al., (2003)Virology 311:275-288: McLellan et al., (2010) J Virology84(23):12236-12244; and McLellan et al., (2011) J Mol Biol 409:853-866).The epitope of 58C5 is located on a domain that goes through the mostdramatic conformational change of the F protein when it moves fromprefusion to the postfusion state. Only the clustered region on thepre-fusion spike agrees with the footprint of an antibody. Therefore,the location of the epitope agrees with data that 58C5 is specific forthe prefusion conformation and it also confirms lack of competition withMotivizumab, F101 and 30D8. Thus, 58C5 does not bind the same epitope asMotivizumab or 30D8.

TABLE 36 Screening of 58C5 with Library including first miniprep 58C530D8 Polyclonal 58C5 30D8 Ratio S1 S2 S3 S1 S2 S3 S1 Mean Mean Mutation30D8/58C5 7.6 4.8 4.8 50.7 55.7 64.7 5.8 53.2 K168E 9.2 13.2 4.8 4.622.6 20.8 27.1 68.0 7.5 23.5 N63H 3.1 22.4 10.0 11.0 17.5 27.9 36.6 60.514.5 27.3 V207D 1.9 50.7 30.7 33.9 59.8 87.9 80.0 82.3 38.4 75.9 S405A2.0 5.4 1.6 3.6 9.7 20.7 12.5 18.9 3.6 14.3 E82G 4.0

TABLE 37 Screening of 58C5 with Library including second miniprepAntibody 58C5 S1 8.6 55.6 13.1 11.1 S2 8.9 16.1 10.2 7.2 S3 7.9 25.310.2 6.3 S4 2.1 54.5 1.2 2.4 30D8 S1 35.7 74.9 9.3 17.2 S2 12 111.1 42.112.1 S3 12 52.3 36.6 12.1 S4 15.2 80 2.4 19.7 Motivizumab Mean 42.1100.9 26.9 24.8 Polyclonal S1 37.1 137.1 14.4 7.7 58C5 Mean 6.9 37.9 8.76.8 30D8 Mean 18.7 79.6 22.6 15.3 Mutation E82G E463A N371D Y86C Ratio30D8/58C5 2.7 2.1 2.6 2.3

2. Binding to Antibody 30D8

The results are shown in Tables 38-39 below, which set forth theimmunoluminescence for at least three samples (S1, S2 and S3) forbinding of 58C5 and 30D8 to each F protein mutant, binding ofmotavizumab and/or the polyclonal serum, the mean of the values for 58C5and 30D8, the ratio of binding to 58C5/30D8 and the mutation in the Fprotein. As shown in Tables 38-39 below, mutation of residues V278,R339, F351, P353, M370, N371, C382, D392, M396 and/or 5403 in the Fprotein resulted in reduced binding of the antibody 30D8. Residue R339was identified in both miniprep screens. Two different mutations of bothM370 and C382 resulted in a reduced binding of antibody 30D8 to the RSVF protein. As shown above for 58C5, the identified residues arerestricted to one domain of the RSV F protein. When all identifiedresidues for binding are combined, they all cluster in the D1 domain, atthe base of the lollipop just above the stem/stalk region. Some residuesare internal according to the 3D model, whereas other residues that areexposed cluster in the interface in the crevice between D1 domains ofdifferent protomers in the prefusion trimer model (see FIG. 3). When theresidues are displayed in the postfusion trimer, all identified residuesare internal. Although the conformational change of the D1 region isexpected to be very limited between pre and postfusion conformations,the protomer subunits do rearrange between pre and post fusion,resulting in a postfusion structure that is more condensed and lessopen. The mapping of the 30D8 Mab to the D1 region agrees with ourresults that it is dependent on the RSV F protein structuralconformation. Moreover, just as for 58C5, the epitope does not competewith 58C5, Motivizumab or antibody 101F. This agrees with the locationon the 3D model. Thus, 30D8 does not bind the same epitope asMotivizumab or 58C5.

TABLE 38 Screening of 30D8 with Library including first miniprepAntibody 58C5 S1 28.3 10 32.4 18.1 13.8 2.9 S2 S3 28.7 23.5 31.5 43.226.4 6.1 S4 44.1 13.9 32.7 26.3 13.2 14.7 30D8 S1 16.8 7.3 20.1 14.1 9.31.4 S2 6.6 4.9 3.3 10.7 3.1 −1.4 S3 1.9 1.1 2.3 7.2 1.5 −1.3 S4 12.211.3 23.7 17.2 19.4 5 Motivizumab Mean 28 Polyclonal S1 24.8 76.3 3 75.913.1 3.8 58C5 Mean 32.4 17.7 33.8 32.7 19.9 7.4 30D8 Mean 9.4 6.2 12.412.3 8.3 0.9 Mutations C382R R339W S403A P353S C382S F351S Ratio58C5/30D8 3.5 2.9 2.7 2.7 2.4 8.2

TABLE 39 Screening of 30D8 with Library including second miniprep 58C530D8 Polyclonal 58C5 30D8 Ratio S1 S2 S3 S1 S2 S3 S1 Mean Mean Mutations58C5/30D8 27.1 29.0 41.7 10.3 9.0 3.6 118.7 32.6 7.6 R339Q 4.3 26.0 37.643.5 16.8 3.1 9.0 61.2 35.7 9.7 M370R 3.7 93.0 47.6 46.1 38.8 10.1 6.735.2 62.2 18.5 M370T 3.4 45.2 66.4 133.6 46.6 11.8 16.6 69.1 81.7 25.0N371Y 3.3 13.0 39.4 43.9 15.3 8.3 6.4 73.5 32.1 10.0 M396V 3.2 86.1 51.263.6 44.0 11.2 10.3 66.8 67.0 21.9 D392N 3.1 122.1 97.6 79.6 63.6 21.011.6 57.5 99.8 32.1 V278M 3.1

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

What is claimed is:
 1. A chimeric anti-respiratory syncytial virus (RSV)antibody or antigen-binding fragment thereof, comprising: a heavy chainCDR1 of SEQ ID NO: 458, a heavy chain CDR2 of SEQ ID NO: 459, a heavychain CDR3 of SEQ ID NO: 460; a light chain CDR1 of SEQ ID NO: 461, alight chain CDR2 of SEQ ID NO: 462, and a light chain CDR3 of SEQ ID NO:463.
 2. The chimeric anti-RSV antibody of claim 1, wherein the chimericanti-RSV antibody is a full-length antibody.
 3. The chimeric anti-RSVantibody of claim 1, wherein the chimeric anti-RSV antibody is afull-length IgG antibody.
 4. The chimeric anti-RSV antibody orantigen-binding fragment of claim 1, wherein the chimeric anti-RSVantibody or antigen-binding fragment is an antigen-binding fragmentselected from the group consisting of a Fab, Fab′, F(ab′)₂, single-chainFv (scFv), Fv, dsFv, diabody, Fd and Fd′ fragments.
 5. The chimericanti-RSV antibody or antigen-binding fragment of claim 1, wherein thechimeric anti-RSV antibody or antigen-binding fragment is a Fab or scFv.6. The chimeric antibody or antigen-binding fragment of claim 1, whereinthe chimeric anti-RSV antibody or antigen-binding fragment neutralizesRSV.
 7. The chimeric antibody or antigen-binding fragment of claim 1,wherein the chimeric anti-RSV antibody or antigen-binding fragmentneutralizes RSV subtypes A and B.
 8. A multivalent antibody comprising:a first antigen-binding portion comprising the chimeric antibody orantigen-binding fragment of claim 1 or such an antigen-binding fragmentthereof conjugated to a multimerization domain; and a secondantigen-binding portion comprising an antigen-binding fragment of anantiviral antibody conjugated to a second multimerization domain,wherein: the first multimerization domain and the second multimerizationdomain are complementary or the same, whereby the first antigen-bindingportion and second antigen-binding portion form a multivalent antibody.9. The multivalent antibody of claim 8, wherein the secondantigen-binding portion comprises an anti-RSV antibody orantigen-binding fragment thereof.
 10. The multivalent antibody of claim8, wherein the anti-RSV antibody or antigen-binding fragment is anantibody or antigen-binding fragment, wherein RSV does not produce avirus that escapes neutralization by the antibody or antigen-bindingfragment thereof after more than 10 or more rounds of viral replicationin the presence of the antibody or antigen-binding fragment thereof. 11.A pharmaceutical composition comprising: the antibody or antigen-bindingfragment of claim 1; and a pharmaceutically acceptable carrier orexcipient.
 12. The pharmaceutical composition of claim 11 formulated asa gel, ointment, liquid, suspension, aerosol, tablet, pill or powder.13. The pharmaceutical composition of claim 11 formulated as a nasalspray.
 14. The pharmaceutical composition of claim 11 formulated forpulmonary, intranasal, or parenteral administration.
 15. Thepharmaceutical composition of claim 11 formulated for single dosageadministration.
 16. The pharmaceutical composition of claim 11 that is asustained release formulation.
 17. A method of treating a viralinfection in a subject, the method comprising: administering to thesubject a therapeutically effective amount of the pharmaceuticalcomposition of claim
 11. 18. A method of treating or inhibiting one ormore symptoms of a viral infection in a subject, the method comprising:administering to the subject a therapeutically effective amount of thepharmaceutical composition of claim
 11. 19. A method of inhibiting aviral infection in a subject, the method comprising: administering tothe subject a prophylactically effective amount of the pharmaceuticalcomposition of claim
 11. 20. The method of claim 17, wherein the viralinfection is an RSV infection.
 21. The method of claim 20, wherein theinfection is an upper respiratory tract infection.
 22. The method ofclaim 17, wherein the subject is a mammal.
 23. The method of claim 22,wherein the subject is a human.
 24. The method of claim 23, wherein thehuman subject is a human infant, a human infant born prematurely or atrisk of hospitalization for a RSV infection, an elderly human, a humansubject who has cystic fibrosis, bronchopulmonary dysplasia, congenitalheart disease, congenital immunodeficiency, acquired immunodeficiency,leukemia, or non-Hodgkin lymphoma or a human subject having an organ ortissue transplant or a blood transfusion.
 25. The method of claim 24,wherein the transplant is a bone marrow transplant or a livertransplant.
 26. The method of claim 17, wherein the pharmaceuticalcomposition is administered topically, parenterally, locally, orsystemically.
 27. The method of claim 17, wherein the pharmaceuticalcomposition is administered intranasally, intramuscularly,intradermally, intraperitoneally, intravenously, subcutaneously, orally,or by pulmonary administration.
 28. The method of claim 17, wherein thecomposition is administered by a nebulizer or an inhaler.
 29. The methodof claim 17, wherein the composition is administered one time, twotimes, three times, four times or five times during RSV season.
 30. Themethod of claim 17, wherein the composition is administered one time,two times, three times, four times or five times within one month, twomonths or three months, prior to a RSV season.
 31. The method of claim17, further comprising administration of one or more antiviral agents.32. The method of claim 31, wherein the antiviral agent is ribavirin.33. The method of claim 17, further comprising administration of one ormore additional antiviral antibodies or antigen-binding fragmentsthereof.
 34. The method of claim 33, wherein the one or more additionalantiviral antibodies are anti-RSV antibodies or antigen-bindingfragments thereof.
 35. The method of claim 34, wherein the one or moreadditional anti-RSV antibodies are selected from the group consisting ofpalivizumab, motavizumab, AFFF, P12f2, P12f4, P11d4, Ale9, A12a6, A13c4,A17d4, A4B4, A8c7, 1X-493L1, FR H3-3F4, M3H9, Y10H6, DG, AFFF(1), 6H8,L1-7E5, L2-15B10, A13a11, A1h5, A4B4(1), A4B4LIFR-S28R, A4B4-F52S, rsv6,rsvl 1, rsvl 3, rsvl 9, rsv21, rsv22, rsv23, RF-1, RF-2, andantigen-binding fragments thereof.
 36. The method of claim 33, whereinthe one or more additional antiviral antibodies is selected from thegroup consisting of an antibody or antigen-binding fragment thatimmunospecifically binds an antigen of parainfluenza virus (PIV) orhuman metapneumovirus (hMPV).
 37. The method of claim 31, wherein thepharmaceutical composition and the antiviral agent are formulated asseparate compositions.
 38. The method of claim 31, wherein thepharmaceutical composition and the antiviral agent are administeredsequentially, simultaneously, or intermittently.
 39. The method of claim17, further comprising administration of hormonal therapy, immunotherapyor an anti-inflammatory agent.
 40. A method of detecting respiratorysyncytial virus (RSV) infection in a subject, the method comprising: (a)assaying the level of RSV antigen in a fluid, cell, or tissue samplefrom the subject utilizing the antibody or antigen-binding fragment ofclaim 1; and (b) comparing the assayed level of RSV antigen with acontrol level wherein an increase in the assayed level of RSV antigencompared to the control level of the RSV antigen is indicative of a RSVinfection.
 41. A polynucleotide encoding the chimeric antibody orantigen-binding fragment of claim
 1. 42. A polynucleotide encoding theheavy chain of the chimeric antibody of claim
 1. 43. A polynucleotideencoding the light chain of the chimeric antibody of claim
 1. 44. A kitcomprising the chimeric antibody or antigen-binding fragment of claim 1,in one or more containers, and instructions for use.